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Enzyme and Microbial Technology 53 (2013) 378– 385

Contents lists available at ScienceDirect

Enzyme and Microbial Technology

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dentification of a laccase from Ganoderma lucidum CBS 229.93 havingotential for enhancing cellulase catalyzed lignocellulose degradation

nna K. Sitarza, Jørn D. Mikkelsena, Peter Højrupb, Anne S. Meyera,∗

Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Building 229, Technical University of Denmark, 2800 Lyngby,enmarkDepartment of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark

r t i c l e i n f o

rticle history:eceived 11 May 2013eceived in revised form 9 August 2013ccepted 16 August 2013

eywords:anoderma lucidumaccaseugar cane bagasseigninative PAGEignocellulose degradation

a b s t r a c t

Based on a differential pre-screening of 44 white-rot fungi on a lignocellulose-supplemented minimalmedium, four basidiomycetes were selected for further study: Ganoderma lucidum, Polyporus brumalis,Polyporus ciliatus and Trametes versicolor. Only G. lucidum was able to grow vividly on malt extract orminimal media supplemented with alkali lignin. When grown on malt extract or minimal medium sup-plemented with lignocellulose (sugar cane bagasse), the crude G. lucidum protein extract exhibited highlaccase activity, ∼3 U/mL toward syringaldazine. This activity was 13–17 fold higher than the corre-sponding activities of the crude protein extracts of P. brumalis, P. ciliatus and T. versicolor. Native PAGEelectrophoresis of the crude G. lucidum extract confirmed the presence of an active laccase. The G. lucidumlaccase had a molecular weight of ∼62.5 kDa, and a Km value of 0.107 mM (determined on ABTS). A par-tial amino acid sequence analysis of four short de novo sequenced peptides, defined after trypsin digestanalysis using MALDI-TOF MS/MS analysis, revealed 64–100% homology to sequences in related laccases

in the UniProt database, but also indicated that certain sequence stretches had low homology. Additionof the laccase-rich G. lucidum broth to lignocellulosic biomass (pretreated sugar cane bagasse) togetherwith a state-of-the-art cellulase enzyme preparation (CellicTMCTec1) produced significantly increasedcellulolytic yields, which were also better than those obtained with a T. versicolor laccase addition, indi-cating that the laccase from G. lucidum has unique properties that may be momentous in lignocellulosicbiomass conversion.

. Introduction

Lignocellulosic biomass such as agro-industrial residues (e.g.traw, sugar cane bagasse, corn stover) and forestry materialsre principally made up of cellulose, hemicellulose, and lignin1]. Conversion of such materials is currently studied all over theorld to allow their utilization in cellulosic bioethanol produc-

ion and biorefinery processes. The lignin constitutes an aromaticiopolymeric mass mainly built of p-hydroxy-phenyl, guaiacyl, andyringyl-type phenylpropane subunits (cinnamyl alcohols) whichre linked together by ether and carbon carbon bonds [2]. In plants,ignin forms a strong hydrophobic meshwork which protects thelant cells from microbial and enzymatic attack and enables waterransport through vessels and tracheids in the plant [2,3]. However,

he presence of lignin in the lignocellulose materials used for cellu-osic biofuel production retards the cellulases during the celluloseydrolysis [1,4].

∗ Corresponding author. Tel.: +45 4525 2800; fax: +45 4593 2906.E-mail address: am@kt.dtu.dk (A.S. Meyer).

141-0229/$ – see front matter © 2013 Elsevier Inc. All rights reserved.ttp://dx.doi.org/10.1016/j.enzmictec.2013.08.003

© 2013 Elsevier Inc. All rights reserved.

White-rot fungi are able to grow on wood and can cause decay ofwoody materials [5]. In addition to the fungal production of cellu-lases and hemicellulases this decay is to a certain extent due tothe fungal production of oxidative extracellular enzymes. Theseenzymes catalyze the modification of lignin via catalytic cleav-age of carbon–carbon bonds and can also catalyze the oxidation oflignin-derived compounds [6]. In contrast, brown-rot fungi can onlyutilize the hemicellulose and cellulose of the plant cell wall, leavingthe lignin essentially undigested, albeit modified by demethylationand oxidation [7]. Ganoderma lucidum (lingzhi) is a representativeof white-rot fungi (Basidiomycota). The fungus has a long historyof use as a remedy in traditional medicine in Asia, and has untilrecently mainly been studied for its potential medical benefits [8,9].However, G. lucidum is highly adaptable to grow on and degrade awide variety of agro-industrial lignocellulosic biomass materialsincluding wood. This proficiency is due to the fungal synthesis andpossible secretion of relevant hydrolytic (cellulases and hemicellu-

lases) and oxidative enzymes – of which some of the latter may beable to catalyze oxidative modification of lignin [10]. Recently, thegenome of G. lucidum was sequenced, producing a 43.3 Mb genomeencoding “one of the richest sets of wood degradation enzymes

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mong all of the sequenced basidiomycetes” [11]; moreover, theresumed lignocellulose-related secretome proteins of G. lucidumave recently been qualitatively evaluated based on plate assaycreening and protein analysis [12].

The expression system of oxidative enzymes of white-rot fungis highly regulated by the nutrients that the fungi are exposedo [13]. On this basis, the main tenet of this study was that if

fungus can grow on a medium supplemented with lignin, itay produce extracellular enzyme(s) that can promote degrada-

ion/modification of lignin or lignin-derived components. Secondly,f such enzyme activity would be supplemented to a lignocellu-osic substrate undergoing cellulolytic hydrolysis, then the addednzyme activity might boost the conversion of the cellulose.ccordingly, by assessing the growth of 44 selected white-rot funginown to grow on decaying plant and/or wood material, incl. G.

ucidum, we aimed to:

i) identify a fungus with the ability to grow on a lignocellulosicsubstrate and/or directly on lignin;

ii) discover enzymes possessing the relevant (oxidative) lignin-modifying enzyme activity, and

ii) preliminarily assess whether this enzyme activity in turn couldboost enzymatic cellulose degradation on a genuine lignocel-lulosic substrate, in casu sugar cane bagasse, which consists ofthe residual stalks from sucrose extraction from sugar cane, andwhich has recently gained particular interest as a potentiallyprofitable substrate for cellulosic biofuel production mainly dueto the abundant amounts available in Brazil, Mexico, India, andAustralia [14,15].

Our focus was primarily on laccases because hydrogen peroxideddition, required for peroxidases, is currently not feasible in indus-rial enzymatic lignocellulose conversion. Laccases (EC 1.10.3.2) arelue copper containing enzymes, which catalyze the removal ofn electron and a proton from phenolic hydroxyl groups (or anromatic amino group) to form free phenoxy radicals. During thexidation reaction one molecule of O2 is simultaneously reduced towo molecules of H2O, so that the catalysis is taking place via trans-er of 4 electrons per round of catalysis [16]. Laccases are also able toatalyze the oxidation of non-phenolic lignin units (C4-esterified)o radicals, usually acting via a mediator [17].

The a priori probability for identifying lignin-degrading laccasesrom fungal growth screening on lignocellulose is based on thatome fungi are able to degrade lignin even though they lack thebility to produce lignin peroxidase and manganese peroxidase18].

. Materials and methods

.1. Organisms

40 strains of the genus Alternaria, Fusarium, Memnoniella, Stemphylium, and Ulo-ladium were obtained from the IBT Culture Collection at the Technical University ofenmark (Lyngby, Denmark). The 40 strains were selected based on knowledge of

heir lignocellulosic/plant material habitat. The fungal isolates were listed by theirBT numbers (S1, Supplementary material). In addition, four white-rot fungal strainsf G. lucidum (CBS 229.93), Trametes versicolor (CBS 100.29), Polyporus brumalis (CBS70.72), and Polyporus ciliatus (CBS 366.74), were purchased from the CBS Fungaliodiversity Center (Utrecht, The Netherlands). All cultures were maintained on Maltxtract Agar (MEA) slants at 4◦C.

.2. Substrates

Sugar cane bagasse (SCB) was obtained from the American Society of Sugar Caneechnologists, Florida Division (LaBelle, FL, USA). The biomass was washed in dis-

illed water (5 times) to remove sand particles. The washed SCB was then dried at0 ◦C for 44 h to a dry matter (DM) content of 97% (w/w), milled in a coffee mill,nd sieved to pass a sieve size of 500 �m (Endecotts, London, UK). The raw SCBontained: 51.1% (w/w) cellulose, 23.1% (w/w) hemicellulose, and 21.6% (w/w) acidnsoluble lignin.

l Technology 53 (2013) 378– 385 379

For the enzymatic lignocellulose hydrolysis study, the SCB was pretreated bysteam explosion: 15% dry matter (w/v) at 175 ◦C for 10 min with 11 bars pressureand a double addition of oxygen (3 min each session) as described previously [19].After the steam explosion pretreatment, the filter cake and the hydrolysate weremixed, dried at 55 ◦C for 44 h, and milled using a coffee-mill and sieved to pass asieve size 210 �m (Endecotts, London, UK). The pretreated SCB contained: 43.8%(w/w) cellulose, 13.8% (w/w) hemicellulose, and 19.3% (w/w) insoluble lignin.

The DM content and the biomass composition were determined according tothe NREL procedure [20]. The levels of glucose and xylose liberated after strong acidhydrolysis were determined by HPAEC using a Dionex BioLC system equipped witha Dionex CarboPac PA1 analytical column (Dionex, Sunnyvale, CA, USA) and an elec-trochemical detector used in the pulsed amperiometric detection mode principallyas described previously [21] and as detailed in Ref. [22].

Alkali lignin (AL) was purchased from NacalaiTesque (Kyoto, Japan), Avicel, atradename for microcrystalline acid treated cellulose powder derived from woodpulp (CAS#9004-34-6), was from Sigma–Aldrich (Steinheim, Germany).

2.3. Media

The Malt Extract medium (MEA) contained: malt extract 20 g/L; peptone 1 g/L;glucose 20 g/L, and agar 20 g/L for agar slants. MEA medium was supplemented with1 mL of a stock trace metal solution (1 g/L ZnSO4·7H2O and 0.5 g/L CuSO4·5H2O).

Minimal Medium (MM), was prepared as described before [23] and containedNH4NO3 1 g/L; Na2HPO4 0.2 g/L; KH2PO4 0.8 g/L; MgSO4·7H2O 0.5 g/L. For the differ-ential pre-screening of the 44 fungal strains, the MM medium was supplementedwith 10 g/L of SCB and 2 g/L of glucose (when 10 g/L of SCB was added).

For the subsequent growth evaluation of the 4 selected white-rot fungi (G.lucidum, T. versicolor, P. brumalis, P. ciliatus), the following media were used: MEAmedium supplemented with 10 g/L SCB; MM medium and MEA medium each sup-plemented with a combination of 5 g/L SCB and 5 g/L Avicel; MM medium and MEAmedium each supplemented with 10 g/L AL (the MM medium was additionally sup-plemented with 2 g/L of glucose when 10 g/L AL was added). The pH of all mediawas adjusted to 5.6 with 2 M NaOH prior to autoclavation, since the native pH of theMEA medium, pH ∼4.7, is considered too acidic for broad screens. A pH of the mediaof pH 5.5–5.6 is recommended and widely used in our lab to ensure growth of fungiin exploratory fungal screenings [24]. All fungal cultures were cultivated at 25 ◦C fora period of 16 and 30 days on the MEA and MM supplemented media, respectively.The growth of the fungi in the liquid media was always performed using a solid,floating support of Leca® beads (Johannes Fog, Lyngby, Denmark). The Leca® beadswere added and autoclaved together with the medium principally as described inRef. [25].

All the media used were inoculated with a 50 mm-diameter disk cut out of themost peripheral side of the MEA agar plate, after a 7-day cultivation of the respectivefungus.

2.4. Freeze-drying of crude fungal protein extracts

After growth in the respective liquid media, the crude fungal protein extractswere obtained after removal of mycelia and Leca® beads by centrifugation at10,000 rpm for 20 min. The crude extract was frozen for 2 h prior to freeze-drying.Freeze-drying of the crude liquid extracts (15 mL) was performed on a Lyovac GT 2(Gea Process Eng. Inc., Columbia, MD) for 2 days until a visible dry pellet was formed.The dry pellet was then concentrated 10 times by solubilization in 1.5 mL of waterand used for the ABTS plate assays, SDS-PAGE and Native-PAGE analysis.

Protein quantification was performed using the Pierce BCA (BiCinchoninic Acid)protein assay kit microplate procedure according to the manufacturer’s instructions(Thermo Fisher Scientific, Rockford, US) as described previously [26]. Bovine SerumAlbumin (BSA) obtained from Sigma-Aldrich (Steinheim, Germany) was used as aprotein standard.

2.5. Enzymes and assay chemicals

Laccase from T. versicolor (Cat. no. 51639), lignin peroxidase (Cat. no. 42603),manganese peroxidase (Cat. no. 93014), syringaldazine, and ABTS (2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) were obtained from Sigma–Aldrich (Stein-heim, Germany). The cellulase preparation CellicTMCTec1, derived from Trichodermareesei, was from Novozymes (Bagsværd, Denmark). Apart from the cellulolyticenzyme base from T. reesei containing at least the two main cellobiohydrolasesEC 3.2.1.91 (Cel6A and Cel7A), five different endo-1,4-�-glucanases EC 3.2.1.4(Cel7B, Cel5A, Cel12A, Cel61A, and Cel45A), �-glucosidase EC 3.2.1.21, and a �-xylosidase [27] the CellicTMCTec1preparation also contains additional �-glucosidaseand particular hydrolysis-boosting proteins [28,29]. The CellicTMCTec1preparationcontained 175 mg protein/mL, 63.8 filter paper units (FPU)/mL and 868 cellobiaseunits (CBU)/mL; the FPU were determined according to the NREL procedure [30],CBU were assessed by the assay of Ghose [31].

2.6. ABTS plate assay for laccase activity visualization

The assay was performed according to the procedure described by Srinivasanet al. [32]. Development of a green halo (positive response for oxidative enzyme

380 A.K. Sitarz et al. / Enzyme and Microbial Technology 53 (2013) 378– 385

Table 1Evaluation of the fungal growtha on the recalcitrant substrate supplemented into both MM and MEA medium, respectively.

Cultivation media usedb Ganoderma lucidum Polyporus brumalis Polyporus ciliatus Trametes versicolor

ALc MM + ± ± ±MEA ++ – – –

SCB + Avid MM ++ + + +MEA +++ + + +

SCBe MM ++ + + +MEA +++ + + +

a The grading of the fungal growth on MM and MEA medium with the supplementations as indicated were graded on a semi-quantitative (+), (–) scale: (±) faint growth(single mycelium colonies), (+) good growth (mycelium mat covering the whole diameter of the experimental tube), (++) better growth (as in case of good growth butmycelium mat thicker), (+++) exceptional growth (thick mycelium mat plus spreading fungal growth on the walls of the experimental tube), and (–) no growth. The gradingof the fungal growth was based on the area of mycelium (colony size) floating on the Leca® beads support (see Section 2) added to the cultivation medium.

b Cultivation media used in this study; MEA (malt extract medium) and MM (minimal medium).vicelvicelvicel

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ctivity) was monitored after adding 25 �L of a 10× concentrated crude extract solu-ion (resuspended in water as described above) into a 4 mm well in the ABTS-agarlate. 25 �L of boiled crude extract solution was used as a negative control, while5 �L of commercial preparations of laccase (from T. versicolor), lignin peroxidase,nd manganese peroxidase were used as enzyme activity controls and were dilutedo reach the same concentration of U/mL, prior to the ABTS-plate assay. The potentialalse-positive effect of ABTS being oxidized by other than laccase factors/enzymesresent in the crude protein extract was evaluated by addition of H2O2 (final con-entration of 0.003%, w/v) to all commercial protein preparations, and by additionf pure H2O2 (final concentrations of 30, 0.3, 0.03 and 0.003%, w/v) to the well ofhe ABTS plate.

.7. Laccase activity assay

Laccase activity was measured spectrophotometrically using syringaldazines substrate, based on the method of Ride [33] as well as the recommendedyringaldazine-based assay protocol for laccase provided by Sigma–Aldrich (Stein-eim, Germany) supplying the syringaldazine [34]. Laccase activity was defineds the amount of enzyme required to oxidize 1 �mol of syringaldazine perinute at 30 ◦C and pH 6.5. Oxidation of syringaldazine was monitored at

30 nm (ε530 = 65,000 M−1cm−1, [35]) for 10 min. The assay mixture contained:00 mM phosphate buffer (2.2 mL, pH 6.5), syringaldazine (0.3 mL, 0.216 mM)nd a pre-diluted fungal crude extract (0.5 mL) that ensured the linear range ofichaelis–Menten kinetics.

The pre-diluted fungal crude extracts were filtered through a 0.45 �m filterMiniSart-plus, sterile, Sartorius, Germany) prior to activity measurements.

.8. SDS-PAGE

The yield of expressed proteins in the fungal crude extract was evaluated byodium Dodecyl Sulphate polyacrylamid electrophoresis (SDS-PAGE), using a Cri-erion XT gel system (Bio-Rad, CA, USA). The protein samples (65 �L) were dilutedn XT sample buffer (25 �L, cat. no. 161-0791) and 500 mM dithiothreitol (10 �L,igma–Aldrich, Germany). The samples were boiled at 95◦C for 5 min before beingoaded into a 10% separation gel (cat. no. 345-0118). Electrophoresis was carried outt a constant voltage of 125 V for 2 h using 5 times diluted XT MOPS as the runninguffer (cat. no. 161-0788). The separated proteins were visualized by staining withoomassie Blue G-250 (cat. no. 161-0786). Estimation of molecular weights (MW)f the proteins was made against molecular (stained) standards (250, 150, 100, 75,0, 37, 25, 20, 15, 10 kDa) (cat. no. 161-0374). All chemicals used for SDS-PAGE wereurchased from Bio-Rad, CA, USA.

.9. Native PAGE

In order to assign the activity of protein at ∼62.5 kDa to laccase, a native PAGElectrophoresis was performed and proteins in the gel were stained with a laccasepecific substrate (1,8-diaminonaphthalene, DAN). Freeze-dried supernatant sam-les were mixed in a ratio 1:1 with XT sample buffer, containing no reducing agents,nd loaded onto a Zymogram gel (cat. no. 345-0080) without thermal denaturation.unning buffer and protein standards were the same as for the SDS-PAGE elec-rophoresis. The separated proteins were visualized by incubation of the gel in a0 mM sodium acetate buffer (pH 5) containing dimethyl sulfoxide (1%) and DANolution (2 mM), as detailed in Ref. [36].

.10. Kinetic studies of laccase isolated from G. lucidum’s cultivation broth

The Michaelis constant (Km) was calculated from a Hanes plot. The substrateas ABTS at final concentrations of: 0.1–11.3 mM [37]. The conditions used for the

nzymatic reaction were as detailed in Ref. [38].

(SCB + Avi), and sugar cane bagasse (SCB), respectively. (SCB + Avi), and sugar cane bagasse (SCB), respectively. (SCB + Avi), and sugar cane bagasse (SCB), respectively.

2.11. MALDI Q TOF MS/MS analysis and conformation of laccase sequence

The band of interest (∼62.5 kDa) was excised from SDS-PAGE gel and in-geldigested with trypsin prior to MALDI-TOF analysis as previously described [39]and as detailed in Ref. [40]. The trypsin digestion and sample analysis were carriedout at the Dept. of Biochemistry and Molecular Biology at the University of South-ern Denmark (Odense, Denmark). The short amino acid peptide sequences wereobtained by the de novo sequencing and were analyzed using 4800 ProteomicsAnalyzer (Applied Biosystems, Foster City, CA, USA) in MS/MS mode, followed bymanual interpretation of the obtained MS/MS spectra by the AminoCalc program(Protana, Odense, Denmark).

2.12. Enzymatic degradation of pretreated SCB during laccase and cellulasecatalysis

Enzymatic lignocellulose degradation was assessed on 0.8% (w/v (DM)) pre-treated SCB suspended in 50 mM acetate buffer (pH 4.8) at 50 ◦C with CellicTMCTec1treatment at a final protein concentration of 80 �g/mL as a base. The crude proteinextract of G. lucidum was spiked onto the CellicTMCTec1 base at a protein concen-tration of 12.3 �g/mL; similarly, the commercial T. versicolor laccase control wasadded at a protein concentration of 12.3 �g/mL. The extent of lignocellulosic biomassdegradation was monitored by sampling from the enzymatic hydrolysis reactionsat 0, 1, 3, 5, and 24 h; each sample was filtered immediately using a 0.2 �m syringetip filter, and mixed directly with the alkaline color reagent solution (see below)and the mixture was incubated at 70 ◦C for 10 min to allow for determination ofreducing ends according to a modified, down-scaled method [41] based on Lever’soriginal colorimetric method with 4-hydroxybenzoic acid hydrazide (Pahbah) [42].The color reagent for the reducing ends assessment was prepared by mixing 1 Mbismuth nitrate, 1 M potassium sodium tartrate and 3 M sodium hydroxide with0.5 M sodium hydroxide and 5% (w/v) Pahbah in 0.5 M hydrochloric acid in the ratio1:899:100 [41]. Glucose was used as standard. All determinations on the enzymatichydrolysis samples were done minimum in duplicate, and data are given as averagevalues in glucose equivalents (mM).

3. Results

3.1. White-rot fungal growth screening

44 white-rot fungal isolates belonging to the phyla Ascomycota(strains of the genus Alternaria, Fusarium, Memnoniella, Stemphylim,and Ulocladium (S1, Supplementary material)) and Basidiomy-cota (strains of the genus Ganoderma, Trametes, and Polyporus),were pre-screened for growth on minimal medium (MM) sup-plemented with sugar cane bagasse (SCB) used as an example ofa genuine lignocellulosic substrate. The result of this differentialpre-screening showed that only four of the basidiomycete isolates,namely G. lucidum, T. versicolor, P. brumalis, and P. ciliatus were ableto grow on the SCB medium (within a 2 week growth period). Inorder to further evaluate the possible ability of any of these four

strains to exhibit growth on lignin – and then implicitly expresslignin-modifying or degrading enzymes – these four strains wereinoculated on MM and MEA medium each supplemented with alkalilignin (AL) and with SCB + Avicel, respectively.

A.K. Sitarz et al. / Enzyme and Microbial Technology 53 (2013) 378– 385 381

Table 2Kinetic constants of laccases and the pH value at which Km was measured.

Substrate Laccase Km (mM) pH Reference

ABTS Pleurotus sajor-caju Lac4 2.50 3.3 [62]Myceliophthora thermophila Lcc1 0.29 6 [63]Pleurotus ostreatus POXC 0.28 3 [64]Pleurotus ostreatus POXA2 0.12 3 [64]Ganoderma lucidum CBS 229.93 0.107 5 this articlePleurotus ostreatus POXA1 0.09 3 [64]Rhizoctonia solani Lcc4 0.052 5.3 [65]

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Fig. 1. A comparison of the laccase activity in the crude protein extracts from G.lucidum, P. ciliatus, P. brumalis, and T. versicolor, cultivated on MEA and MM media,supplemented with SCB or SCB + Av, where SCB – sugar cane bagasse, and Av – Avicel.The highest activity of laccase was observed for the crude protein extracts from G.lucidum. In general, laccase activity was higher when fungal strains were cultivatedon MEA (SCB and SCB + Av) media. The activity of laccase for T. versicolor could not

Ganoderma lucidum GaLc3 0.037 5 [43]Trametes trogii POXL3 0.03 3.4 [66]

Out of the four basidiomycetes fungi tested, only G. lucidumxhibited significant growth on both media supplemented with ALTable 1). P. brumalis, P. ciliatus and T. versicolor only produced aaint mycelial growth on the MM medium supplemented with ALnd they were unable to grow on the MEA medium when AL wasdded (Table 1). The G. lucidum strain also grew well on the MMedium with SCB + Avicel and exceptionally better on the MEAith SCB + Avicel. Its growth was vivid and the colony size wasuch bigger (in diameter) and thicker when compared to that of

. brumalis, P. ciliatus and the well-studied oxidative enzyme pro-ucer – T. versicolor (Table 1).

.2. Oxidative enzyme activity assessment of the fungal cruderotein extracts on ABTS

ABTS oxidation was used to evaluate the possible presence ofxidative enzyme activity in the crude fungal extracts: The oxi-ation of ABTS in the agar plate assay followed the growth abilityattern on the different media tested, meaning that if a fungus couldrow on a medium supplemented with SCB (with or without Avi-el) or AL, then it also expressed enzymes that enabled activity onBTS. The strongest response in the ABTS plate assay (appearancef a green halo, data not shown) was observed for the crude proteinxtract of G. lucidum for all media tested. No response for oxidationf ABTS was observed for P. brumalis, P. ciliatus and T. versicolor forhe MEA medium supplemented with alkali lignin, which was inccordance with their lack of growth on this medium (Table 1).

The oxidation level of ABTS for the AL supplemented MMedium was very low for P. brumalis, P. ciliatus and T. versicolor

nd in contrast significant for G. lucidum, also in accordance withhe growth ability data (Table 1).

To assure that the ABTS oxidation was not a “false-positive”uto-oxidative response of ABTS, the addition of hydrogen perox-de in concentrations of 30, 0.3, 0.03 and 0.003% (w/v) were tested.nly the H2O2 at a level as high as 30% (w/v) was able to auto-xidize ABTS (data not shown). That result excluded the possibilityhat the oxidation of ABTS was caused by H2O2 produced by fun-al oxidases. Moreover, neither lignin peroxidase nor manganeseeroxidase added, were able to oxidize ABTS unless 0.003% (w/v)2O2 was added to the extract mixture (data not shown).

.3. Kinetic studies of laccase present in the crude extract of G.ucidum

The kinetic studies for the G. lucidum laccase present in therude protein extract were performed using ABTS as a substrate.BTS was used in order to compare the obtained Km value with

hose described in the literature. From the Hanes–Wolf plot, whichave a good distribution of the data points, the Km value was calcu-

ated to be 0.107 mM (Table 2). This value of Km places laccase from. lucidum in the middle range of laccases (presented in Table 2),owever the value obtained is significantly higher than a Km valueelonging to another G. lucidum laccase, namely GaLc3 [43]. The

be determined. The gray and black bars indicate the amount of the laccase activitypresent in the fungal crude protein extracts from MEA and MM media, respectively;vertical bars indicate ± standard deviation.

Vmax value was not calculated due to lack of purity of the analyzedlaccase containing sample.

3.4. Laccase activity assessment for fungal crude protein extractson syringaldazine

The highest activity of laccase present in the fungal crudeprotein extracts was ∼3 U/mL assessed on syringaldazine asassay substrate and was obtained for G. lucidum cultivated onMEA medium supplemented with SCB (3.1 U/mL) or SCB + Avicel(2.9 U/mL), respectively (Fig. 1).

This level of laccase activity was approximately 3.5 times higherthan when G. lucidum was grown on MM medium under the samesupplement conditions (Fig. 1). These activity levels of laccase wereconsistent with the vivid growth of G. lucidum on the media supple-mented with SCB. The laccase activities in the crude extracts fromP. brumalis, P. ciliatus, and T. versicolor, respectively, were 13–17times lower than that obtained for G. lucidum cultivated on the samemedia (i.e. MM or MEA supplemented with SCB or SCB + Avicel)(Fig. 1). Unfortunately, it was not possible to measure laccase activ-ity on media supplemented with AL, due to the interference fromthe black coloring of lignin and inability to separate it from thecultivation media.

3.5. Separation of crude protein extract from G. lucidum usingSDS-PAGE and Native PAGE

The crude G. lucidum protein extract was analyzed by SDS-PAGE.Comparison of bands and MW of a control laccase from T. versicolorwith the proteins from the crude extract suggested the presence ofa laccase (Fig. 2). The band in the separated crude protein extract,corresponding to the laccase from T. versicolor, was approximately62.5 kDa. Additionally, a sharp, distinct band of ∼62.5 kDa was vis-ible in the sample of the MEA medium with AL, regardless of theextensive, black background smearing.

In order to assign that the MW of the protein from G. lucidumbelonged to laccase an in-gel activity staining was performed withthe substrate specific for laccase (1.8-diaminonaphthalene, DAN).

Only a single band at 62.5 kDa in each lane (Fig. 3) was stainedwith DAN. A specific dark brown band was observed which con-firmed presence of an active laccase in the G. lucidum crude extractpreparations derived from the different MEA supplemented media

382 A.K. Sitarz et al. / Enzyme and Microbial Technology 53 (2013) 378– 385

Fig. 2. An electrophoretic separation (SDS-PAGE under denaturating conditions)of the proteins present in the G. lucidum’s crude extract, obtained under differentcultivation conditions. All protein bands were stained with Coomassie Blue G-250.Wells in the gel represent; protein marker (M), laccase protein standard (4), andpam

(fhwdm

3a

eapaodtftdrim

Fig. 3. An electrophoretic separation (Native-PAGE under non-denaturating con-ditions) of the proteins present in the G. lucidum’s crude extract, obtained underdifferent cultivation conditions. All protein bands were stained with Coomassie BlueG-250. Wells in the gel represent; protein marker (M), laccase protein standard (4),

TO

A

rotein crude extract from MEA medium supplemented with SCB (1), SCB + AV (2),nd AL (3). The laccase from G. lucidum (estimated to 62.5 kDa) had a slightly lowerolecular weight than that from a control (T. versicolor, Sigma–Aldrich, Germany).

MEA + SCB; MEA + SCB + Avicel; MEA + AL) (Fig. 3). The extractsrom the MEA supplemented media were selected due to theirigher laccase activity (Fig. 1). Additionally, no color developmentas observed neither for the commercial controls of lignin peroxi-ase and manganese peroxidase (data not shown), nor for the lowerolecular weight proteins from the crude protein extract.

.6. Partial amino acid sequence of laccase by MALDI-TOF/MSnalysis

The protein band at 62.5 kDa in the G. lucidum crude proteinxtract, putatively identified as the laccase enzyme, was furthernalyzed by MS after an in-gel digestion with trypsin. The shorteptide amino acid sequences discovered de novo by MALDI-TOFnalysis had a high homology (64–100%) to laccase sequences fromther basidiomycetes (Table 3). The blasting results in the UniProtatabase [44] showed that the amino acid sequences between posi-ion 88–100 and position 185–197 are highly conserved within theamily of Ganodermataceae. The blasting results thus corroboratedhat the G. lucidum enzyme was a laccase. However, the fact that

ifferent peptide fragments, especially in the amino acid sequenceegions of 245–263 and 452–466, exhibited less than 90% sequencedentity to existing functional laccases implied that the G. lucidum

ight harbor unique sequential features.

able 3verview of the identity of four glycopeptides analyzed by MALDI-TOF from G. lucidum C

UniProt identifier Identified organism Aa seq. Sequence identity of disco

88TTSIHWHGFFQK100 18

Q9GH17 Ganoderma lucidum 7071-9 520 100 1Q9HDS8 Polyporus ciliatus 524 90 1C5HL41 Ganoderma lucidum TR6 520 100 1B5G552 Ganoderma lucidum RZ 520 100 1Q308Q9 Trametes versicolor 522 100

A3F8Z8 Polyporus brumalis lac1 520 100

A3F8Z8 Polyporus brumalis lac2 524 90

Q9UVQ2 Pycnoporus cinnabarinus lac1 518 90

a seq.: amino acid sequence; n.a.: not annotated.

and protein crude extract from MEA medium supplemented with SCB (1), SCB + AV(2), and AL (3). Native PAGE electrophoresis confirmed presence of active laccase at62.5 kDa.

3.7. Enzymatic degradation of lignocellulose (pretreated SCB)

The enzymatic lignocellulose degradation increased signif-icantly beyond the CellicTMCtec1 baseline yield when thelaccase-rich G. lucidum protein extract was added together withCellicTMCtec1 onto lignocellulosic biomass (pretreated SCB) (Fig. 4).Hence, after 24 h of enzymatic treatment, the increase in glucoseequivalents (mM) based on reducing ends measurement repre-sented a ∼75% improvement compared to the corresponding neatCellicTMCtec1 yield (Fig. 4). That the effect was a result of the addedlaccase activity was corroborated by the finding that the G. lucidumprotein extract exhibited no cellulase activity on AZCL-cellulose(data not shown). In contrast, the spiking with the T. versicolor lac-case “positive” control enzyme tended to produce an inhibitoryeffect on the CellicTMCtec1 catalyzed lignocellulose degradation.The exact mechanism behind this effect is uncertain at present.

4. Discussion and conclusions

The initial differential screening showed that only four out of 44

white rot fungi could grow significantly on media supplementedwith lignocellulose in the form of raw sugar cane bagasse. Thefungi that were capable of growing on the SCB and the alkalilignin were hypothesized to potentially express new interesting

BS229.93 to other laccases deposited in the UniProt database.

vered glycopeptides (%)

5GSDSTLINGLGR197 245DDDSTVLTLADWYHVAAR263 452TLSNADIAPDGFTR466

00 80 8100 80 8100 80 8100 75 8893 85 6493 90 7093 n.a. n.a.93 75 68

A.K. Sitarz et al. / Enzyme and Microbia

Fig. 4. A graphical comparison of yields of glucose equivalents (mM) (based onreducing ends measurement) released during 24 h enzymatic treatment of 0.8% (w/v(DM)) steam exploded SCB at pH 4.8 and 50 ◦C. CellicTMCTec1 0 indicates addition ofCellicTMCTec1only; CellicTMCTec1 Tv indicates spiking of CellicTMCTec1 with com-mercially obtained laccase from T. versicolor, and CellicTMCTec1 Gl indicates spikingof CellicTMCTec1with a crude laccase-active extract of G. lucidum (grown on MEAwith SCB). Data are shown as averages ± standard deviation (s.d.); different super-so

elsdbaltaiMwGfdoowttkafafeam[llcrnlceHip

for the growth ability (Table 3) revealed significant differences

cript letters indicate significantly different levels for each time point based onne-way ANOVA (p < 0.05). Pooled standard deviations ranged from 0.04 to 0.26.

nzymes that might be able to catalyze modification of lignin inignocellulosic biomass undergoing cellulosic hydrolysis. In turn,uch lignin modification would yield a higher cellulose conversionuring cellulase catalyzed hydrolysis of lignocellulosic substratesy increasing the cellulase access to the cellulose via expanding thevailable surface area and/or by diminishing the adsorption of cel-ulases to lignin [45]. The growth screening analysis showed thathe growth of P. brumalis, P. ciliatus, and T. versicolor, which wereble to proliferate on sugar cane bagasse, was apparently inhib-ted by addition of AL to a malt extract medium (regardless of the

EA being a sugar and nutrient rich medium). However, G. lucidumas found to grow significantly on the medium with AL added, i.e.. lucidum grew faster and to a larger colony size than the other

ungi tested. T. versicolor is a well studied oxidizing enzyme pro-ucer [46,47] and was chosen as a positive control for assessmentf the growth ability on recalcitrant lignocellulose substrates andn AL. It is well known that the growth of T. versicolor (and otherhite rot fungi) as well as the enzyme induction show some varia-

ion depending on media additives [48], but it was surprising thathe growth of T. versicolor was poor on AL. T. versicolor is thusnown as a lignin-degrading white rot fungus producing laccase,nd laccase from T. versicolor has been reported to improve theermentability of lignocellulosic hydrolysates from wood via cat-lytic oxidation of monophenolic compounds and in turn improveermentability more than for example lignin peroxidase [47]. How-ver, previous studies have found that the T. versicolor laccasectivity will catalyze lignin degradation only in the presence of aediator such as 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate)

38]. The good performance of T. versicolor laccase in lignocel-ulosic hydrolysates may thus be due to the presence of naturalaccase mediators in the hydrolysates. The low growth of T. versi-olor as well as the accompanying low T. versicolor laccase activityecorded in the present study might reflect that mediators wereot added to the growth media. Based on the finding that G.

ucidum could grow with AL added to the medium, it was con-eived that the G. lucidum might produce one or more oxidativenzymes able to catalyze the modification of lignin constituents.

ence, the growth ability of G. lucidum on AL was indeed an

ndication that it had a unique (oxidative) enzyme system as com-ared to P. ciliatus, P. brumalis, and T. versicolor; i.e. an enzyme

l Technology 53 (2013) 378– 385 383

system that allowed G. lucidum to degrade/modify lignin or toovercome the toxic/inactivating effect of AL addition under car-bon and nitrogen rich growth conditions (MEA medium). Whencoupling this interpretation to the growth data on MM or MEA sup-plemented with AL, it might be possible that the laccase from G.lucidum had a role in its ability to grow on the AL supplementedmedia.

This comprehension agrees with the recent findings that theG. lucidum genome harbors genes for 36 putative lignin-modifyingoxidoreductases, including a large set of lignin peroxidases alongwith laccases [9]. The data also agree with previous results whichshow that G. lucidum expresses a number of lignin-degradingenzymes, notably laccases, when cultivated on sugar cane bagasse[12] or on wood substrates (poplar, spruce, oak, and pine) [49].Earlier studies have indicated that white-rot fungi (exemplified byT. versicolor as well as e.g. Ganoderma colossum) only produce lac-case in defined media, and laccase and manganese peroxidase incultures grown with poplar [50–52]. Pelaez et al. [10] reported thatlaccase activity was found in 50% of the 68 fungal strains tested, thatlaccase activity was the highest oxidizing enzyme activity found,and that lignin peroxidase activity was not detected in any of the68 fungi tested.

Tour et al. [53] classified white-rot fungi into five groups basedon the oxidative type of enzyme(s) they produce. Laccase was fre-quently a dominant enzyme, indicating its important role in lignindegradation. Laccase, being a phenol oxidase (EC 1.10.3.2), cat-alyzes the oxidation of phenols and phenolic lignin substructuresby electron abstraction. During this reaction, the enzyme is pre-sumed to simultaneously catalyze the formation of radicals thatcan re-polymerize or lead to depolymerization of the lignin macro-molecule [54]. The subsequent radical coupling processes takeplace independently of the laccase catalysis reaction. Laccase canthus induce polymerization as well as depolymerization whereasmanganese peroxidase and lignin peroxidase only catalyze the for-mation of phenoxy radicals [53]. Due to their adaptable redoxpotential laccases may catalyze the oxidation of both phenolic andnon-phenolic lignin derived substrates [55]. The specific laccasefrom G. lucidum was not induced by carbon depletion. This findingindicates that the laccase expression might be induced by trypto-phan or tyrosine present in the malt extract [56]. Eggert et al. [18]identified a tryptophan derivative (4-hydroxyindole) and a trypto-phane derived metabolite (3-hydroxy-2-aminobenzoate) acting asmediators in laccase-catalyzed reactions performed by P. cinnabar-inus. In general, a change in laccase activity corresponding to thetype of media used (i.e. nitrogen and carbon source availability)is a controversial subject [13]. Some authors found that in certainwhite rot fungi the “ligninolytic” enzyme activity increased undernutrient and carbon limiting conditions, whereas others reportedthe opposite tendency [23,48,52].

The ABTS plate assay was used to grade the oxidative enzymeactivity based on the degree of green color development and thespeed with which the oxidation of ABTS took place. Comparingthese results with the initial growth screening (Table 1), the activ-ity of fungal crude protein extracts on ABTS correlated well withthe growth rate. Additionally, the laccase from G. lucidum hadrelatively high value of Km (0.107 mM), indicating a low enzymeaffinity to the substrate. The substrate selectivity, thermal robust-ness, and the response of the laccase activity to reaction conditionsdeserve further study. The activity band staining of enzymes witha phenolic substrate revealed presence of laccase with a MW of∼62.5 kDa which was further confirmed by MALDI-TOF analysis.The short amino acid sequences compared with the fungi tested

between the amino acids in the short stretches. Additionally, thislaccase showed the highest short-peptide similarity to G. lucidum7071-9 (Q9GH17, UniProt identifier).

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84 A.K. Sitarz et al. / Enzyme and Mi

In studies using 13C and 14C labeled lignin model compoundsawai an co-workers [57,58] have shown that laccase (“laccase

II”) from T. versicolor is able to catalyze C C bond cleavage ofntermonomer linkages as well as ring opening of low-molecular

eight phenolic lignin model compounds causing production ofater-soluble degradation products. It is still uncertain which

xact role these reactions may play in the lignin decay processnd lignin metabolism of T. versicolor. However, if the laccaserom G. lucidum can also catalyze C� C� bond cleavage in lignin,his action may effectuate that low-molecular weight lignin-omponents are removed from the ligno-cellulosic material surfaces water-soluble degradation products. In turn, this would allowhe cellulases better access to the cellulose fibers. The data in Fig. 4,n which the laccase from G. lucidum acted more efficiently than the. versicolor laccase control, would then reflect that the G. lucidumaccase acted with a higher efficiency (higher kcat/Km and/or higheredox potential) than the T. versicolor laccase, and consequentlyausing a faster and more extensive solubilization of lignin degra-ation products on the lignin substrate. Another consequence of

gradual removal of lignin-components from the surface of theignocellulosic material could be that the non-productive adsorp-ion of cellulases to lignin would decrease. Such an effect wouldlso produce improved enzymatic cellulose degradation. Alterna-ively, the laccase action may induce enzymatic oxidation of theignin components to less inhibitory compounds for the cellulaseydrolysis. The G. lucidum laccase was tested on pretreated sugarane bagasse as an example of a genuine, abundantly available lig-ocellulosic substrate which is in focus as a possible substrate forellulosic ethanol production [14,15]. Like other grass lignins, thentreated lignin of sugar cane bagasse contains �-O-4 ether bondss the major linkages between the structural units and only rela-ively low amounts of the more condensed �-� and �-1 units [59],hich are more abundant in e.g. wood lignins. On the other hand,

cidic pretreatment, as used here in the form of acidic presoaking19], will catalyze removal of hemicellulose, but tends to aggregateon-solubilized lignin in contrast to alkaline pretreatment whicholubilizes and removes a large part of the lignin [60]. A furtherlucidation of the nature and action mechanisms of the encircled. lucidum laccase will require that it is successfully cloned andxpressed as a mono-component enzyme in a “work-horse” organ-sm. The putative ability of the G. lucidum laccase to acceleratenzyme catalyzed lignocellulose degradation could prove usefuln the bioethanol and the budding cellulosic biorefinery indus-ry, where high yields of fermentable sugars are desired. The datahus support that G. lucidum, in addition to having potential as atherapeutic fungal biofactory” [61], harbors genes able to expressnteresting enzymes for catalyzing lignocellulosic biomass conver-ion.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.enzmictec.2013.8.003.

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