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Ava i l ab l e on l i ne a t www.sc i enced i r ec t . com

www.e l sev i e r . com/ loca te / j p ro t

Secretome analysis of Ganoderma lucidum cultivated insugarcane bagasse

Tamilvendan Manavalana, Arulmani Manavalanb, c,Kalaichelvan P. Thangavelua,⁎, Klaus Heeseb, c,⁎⁎aCentre for Advanced Studies in Botany, University of Madras, Chennai 600 025, Tamil Nadu, IndiabSchool of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, SingaporecInstitute of Advanced Studies, Nanyang Technological University, 60 Nanyang View, Singapore 639673, Singapore

A R T I C L E I N F O

Abbreviations: ITS, internal transcribed spaSDS-PAGE, sodium dodecyl sulfate polyacrylACN, acetonitrile; FA, formic acid; CMC, carboxperoxidase; DMP, 2,6-dimethoxyphenol; BE, bi⁎ Corresponding author. Tel.: +91 44 22202754⁎⁎ Correspondence to: K. Heese, Institute of AdSingapore. Tel.: +65 6316 2848; fax: +65 6791

E-mail addresses: ptkalai2003@yahoo.com

1874-3919/$ – see front matter © 2012 Elseviehttp://dx.doi.org/10.1016/j.jprot.2012.09.004

A B S T R A C T

Article history:Received 7 June 2012Accepted 8 September 2012Available online 20 September 2012

Harmful environmental issues of fossil-fuels and concerns about petroleum supplies havespurred the search for renewable alternative fuels such as biofuel. Agricultural crop residuesrepresent an abundant renewable resource for future biofuel. To be a viable alternative, abiofuel should provide a net energy gain, have environmental benefits, be economicallyfeasible, and should also be producible in large quantities without reducing food supplies.We used these criteria to evaluate the white rot basidiomycota-derived fungus Ganodermalucidum that secretes substantial amounts of hydrolytic and oxidative enzymes useful forthe degradation of lignocellulosic biomass that were not described hitherto. The currentbottleneck of lignocellulosic biofuel production is the hydrolysis of biomass to sugar.To understand the enzymatic hydrolysis of complex biomasses, we cultured G. lucidumwith sugarcane bagasse as substrate and qualitatively analyzed the entire secretome.The secreted lignocellulolytic enzymes were identified by liquid chromatography–tandemmass spectrometry (LC-MS/MS) and diverse enzymes were found, of which several werenovel lignocellulosic biomass hydrolyzing enzymes. We further explored G. lucidum-derivedcellulose, hemicellulose and lignin degrading enzymes as valuable enzymes for the secondgeneration of biofuel obtained from a lignocellulose substrate such as sugarcane bagasse.

© 2012 Elsevier B.V. All rights reserved.

Keywords:Ganoderma lucidumSugarcane bagasseSecretomeBiofuelLignocellulolytic enzyme

1. Introduction

Within the fungi kingdom, the Polyporaceae family-derivedGanoderma lucidum belongs to the Basidiomycota as part of thesubkingdom Dikarya. In Japan, its fruiting body is called Reishior Mannetake, and in China and Korea it is variously called

cer; PDA, potato dextrose aamide gel electrophoresiymethyl cellulose; CMCaological efficiency; CBM, c; fax: +91 44 22352498.vanced Studies, Nanyang3856.(K.P. Thangavelu), klaus

r B.V. All rights reserved

Ling Chu, Ling Chih or Ling Zhi. G. lucidum has been a populartraditional medicine used to treat various human diseasessuch as anti-inflammatory, anti-tumor, or various cardiovas-cular disorders [1]. Besides, the cultivation of G. lucidum onsolid substrates (agricultural waste) has become essential tomeet the increasing demand in the international markets and

gar; cm, centimeter;mm,millimeter; RH, relative humidity; U, units;s; LC-MS/MS, liquid chromatography–tandem mass spectrometry;se, carboxymethyl cellulase; MnP,manganese peroxidase; LiP, ligninarbohydrate binding module.

Technological University, 60 Nanyang View, Singapore 639673,

.heese@rub.de (K. Heese).

.

299J O U R N A L O F P R O T E O M I C S 7 7 ( 2 0 1 2 ) 2 9 8 – 3 0 9

to make it more accessible and affordable [2]. In particular,because the limitation of fossil fuel reserves and its impacton global warming have led to the urgent need of the iden-tification, development and exploitation of novel sustainablerenewable energy resources to encounter the future require-ments. Though there aremany reports on bioethanol, they aremainly from corn, wheat and sugarcane juice or they usedmolasses as substrates for the production of the firstgeneration of biofuel [3,4]. Besides, the current use of foodsources increases the production cost and also affects thefood-chain supply as well as the socioeconomics [5]. Hence,we used a viable sugarcane bagasse as substrate as it hasmuch less impact on the food-chain supply and the socioeco-nomics. A more viable unconventional source is the biocon-version of low cost agricultural wastes into biofuels,simultaneously alleviating global warming and environmen-tal pollution. The plant biomasses include crop residues,hardwood, softwood, herbaceous biomass and cellulosicwastes which all contain cellulose, hemicellulose and ligninthat are virtually the most abundant renewable and inex-haustible carbon resources. This is thus the most favorablefeedstock for the production of the second generation ofbiofuel. Microbial strains belonging to bacterial and fungaldomains secrete lignocellulolytic enzymes and potentiallydegrade cellulose and hemicellulose while highly recalcitrantlignin is depolymerized predominantly by white rotwood-decay fungi such as G. lucidum and others [6–8], thusalso often used for myco-/bioremediation [9].

Among the different plant biomasses, sugarcane bagassehas been used extensively because of its availability and highvolume/low cost production potential. The agricultural resi-due sugarcane bagasse is generally used as a fuel and consistsof water (46–52% w/w), fiber (43–52% w/w, including cellulose(50%), hemicellulose (25%) and lignin (25%)) and relatively smallquantities of soluble solids (2–6% w/w) [10]. The conversion ofsugarcane bagasse into soluble sugar is a crucial limiting stepwhich therefore points to the urgent need of an economicallyviable production of efficient lignocellulolytic enzymes. Sincethis biopolymer can be mineralized by G. lucidum [11], exploringG. lucidum's secretome by a highly sensitive proteomics technol-ogy could reveal novel highly potential lignocellulose hydrolyz-ing enzymes and thus contribute to a better understanding ofthe lignocellulose hydrolysis mechanism. According to previousstudies, G. lucidum-derived enzymes involved in i) cellulosedegradation (endo 1,4-beta glucanase, exo 1,4-beta glucanaseand beta glucosidase), ii) hemicellulase degradation (endo 1,4-beta xylanase, beta xylosidase, alpha-L-arabinofuranosidase,alpha glucuronidase and acetyl xylan esterase) and iii) lignindegradation (lingnin peroxidase (LiP), manganese peroxidase(MnP) and laccase) have been described [12–15].

The different known—gene/DNA and RNA-based—methodsof estimation and identification of enzymes in a complexsecretome have several drawbacks including: reagent crossreactivity, limits in detection sensitivity, lack of RNA stability,choice of primer sets; or such gene expression methods causea high number of false-positive/-negative results [16,17]. How-ever, the identification of novel enzymes involved in cellulose,hemicellulose and lignin degradation by an innovative technol-ogy is still lacking. Recent advances in sensitive proteomicstechnologies offer opportunities to study the entire proteome

of any sample in a single experiment to identify a diverse set ofproteins from a complex biological sample [6]. In the presentstudy, we report for the first time the cultivation of G. lucidumwith sugarcane bagasse alone as substrate—obtaining within40 days a highest biological efficiency of 80±15% for 500 g of drysubstrate.We analyzed theG. lucidum secretome proteins in theused substrate and comprehensively analyzed and identifiedseveral proteins involved in lignocellulose degradation, includ-ing cellulases (10), hemicellulases (2), glycoside hydrolases (4),lignin degrading proteins (10), a protease (1), phosphatases (3),transport proteins (4), hypothetical proteins (10) and severalother proteins when G. lucidum was cultivated in sugarcanebagasse as a lignocellulose substrate.

2. Materials and methods

2.1. Reagents

All reagents usedwere purchased fromSigma-Aldrich (St. Louis,MO, USA) unless otherwise stated.

2.2. Fungal strain and culture conditions

G. lucidum was isolated from a tamarind trunk wood locatedwithin the Indian Institute of Technology Campus (Chennai,Tamil Nadu, India) and identified asG. lucidum by amplifying andsequencing the ITS (Internal Transcribed Spacer) DNAusing ITS1and ITS4 primers according to previous reports [18] (GenBankaccession number FJ982798) by polymerase chain reaction (PCR)and sequenced as described previously [19–23]. Stock cultures ofG. lucidum (strain dTVK1T; submitted to theMicrobial TypeCultureCollection (MTCC) Centre at Chandigarh, India) weremaintainedon the potato dextrose agar (PDA) slant at 4 °C in the dark. Themycelium from the slant was transferred to PDA plates andincubated at 30 °C for 7 days. Mycelial discs from the peripheralregion of actively growing culture were used as inoculum. Theexperimental design included biological and technical replicatesas outlined in Fig. 1.

2.3. Spawn production

The boiled maize (Zea mays) grains were mixed with 1% (w/w) ofCaCO3 in 250 ml Erlenmeyer flasks and sterilized for 20 min at121 °C, cooled and inoculated with an agar plug (0.6 cm diameter)cut from the peripheral portion of a 7-d-old youngmycelium grownon PDAmedium. Bottles were incubated in the dark, at 30 °C.

2.4. Substrate preparation

Fruiting body cultures were carried out in polypropylene bags(diameter 30 cm, length 60 cm). Five hundred grams of driedsugarcane bagasse substrate wasmixed with 1% (w/w) of CaCO3

to buffer the substrate andmoisture contentwas adjusted to 80%by adding the appropriate quantity of distilled water anddispensed into the polypropylene bag. A plastic ring was slippedthrough the open end of the bag and sealed by a cotton plug.Sterilization was carried out at 121 °C for 20 min. After coolingdown to room temperature, a 10% inoculum (spawn (w/w)) wasused for each bag, which was then placed at 30 °C in the dark.

Fig. 1 – Schematic representation of the experimental design showing the biological replicates of G. lucidum when cultivatedwith sugarcane bagasse as lignocellulose substrate and the whole secretome proteins were identified by LC-MS/MS analysis.

300 J O U R N A L O F P R O T E O M I C S 7 7 ( 2 0 1 2 ) 2 9 8 – 3 0 9

When the mycelium colonized the substrate completely within10–15 days, the plugwas removed and the containersweremovedto an environment at 30±2 °C, 90–95% relative humidity (RH) withillumination (fluorescent lamps, 4 μmol photon m−1 s−1) for theinitiation of primordium, for stipe growth 28±2 °C, 70–80% RH andillumination (fluorescent lamps, 6 μmol photon m−1 s−1) and forthe formation of fruiting bodies 30±2 °C, 85–95% RH withillumination (fluorescent lamps, 15 μmol photon m−1 s−1 for12 h/day). After harvesting, the fruit bodies were shade dried,powdered and stored at room temperature for future use. Thespent substratewasused in further studies for secretomeanalyses.

2.5. Cropping period, crop yield and quality traitsassessment

The mature basidiomycota G. lucidum were collected andthe following production and quality traits were registered:A) production (harvested basidiomycota); B) primordia initia-tion (in days from the start of incubation); C) biological efficiency

(BE): the ratio of kg of fresh mushrooms harvested per kg of drysubstrate and expressed as a percentage.

2.6. Secreted protein isolation from the spent substrateand separation by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE)

Sugarcane bagasse substrates colonized by mycelium werecollected at different stages of the solid-state fermentation(5 to 50 days) for the time course studies of the lignocellulolyticenzymes activities, day 40 was used for the secretome analysisand extracellular proteins were extracted from decomposedspent substrates (10 gwetweight) with 50 ml of sodiumacetatebuffer (100 mM, pH 5) for 1 h at 4 °C under slight shaking. Crudeextracts were recovered by filtration through cheese clothand then centrifuged at 8000 g for 30 min at 4 °C (Eppendorf's(Hamburg, Germany) centrifuge model 5810R). Three indepen-dent experiments were carried out and biological replicateswere pooled to minimize biological variation. Protein content

301J O U R N A L O F P R O T E O M I C S 7 7 ( 2 0 1 2 ) 2 9 8 – 3 0 9

was assayed using a 2-DQuantKit following themanufacturer'sinstructionswith the supplied BSA (bovine serumalbumin) as astandard (GE Healthcare, Amersham Biosciences, Amershamplace, Buckinghamshire, UK). Aliquots of extracellular proteinsamples were stored at −80 °C for further one-dimensional gelelectrophoresis experiments. Fifty μg proteins of the pooledbiological replicates were subjected to each lane (total: 3 lanes=3×50=150 μg proteins) of the 12% SDS-PAGE analysis and runfor 120 min at 100 V [24,25].

2.7. In-gel tryptic digestion

Eight horizontal slices were cut across the three lanes ofthe SDS-PAGE gel of G. lucidum supernatant and cut into smallpieces (approximately 1 mm2), dried by vacuum centrifuga-tion, and reduced by submerging the gel pieces in 100 mMammonium bicarbonate solution containing 10 mMDTT for 1 hat 55 °C. Excess DTT/ammonium bicarbonate was removed, andthe same volumeof 100 mMammoniumbicarbonate containing55 mM iodoacetamide was added and incubated for 45 minin the dark. After alkylation, the gel pieces were treated with100 mMammonium bicarbonate and ACN (acetonitrile) sequen-tially and then dried by vacuum centrifuge. To the dried gelpieces, 2 mg of trypsin (Sigma-Aldrich) was added in sufficientammonium bicarbonate solution (100 mM, pH 8.5) to submergethe gel pieces in the solution. Digestion was carried out at 37 °Covernight. The gel was washed once with ammonium bicarbon-ate followedbyACN, and twicewith 5% formic acid (FA) followedby ACN. Peptides were collected from the washings, dried byvacuum centrifugation, and resuspended in 0.1% formic acid(FA) solution for mass spectrometric analysis.

2.8. LC-MS/MS and data search analysis

The in-gel digested peptides were separated and analyzed on aShimadzu UFLC system coupled to an LTQ-FT Ultra (ThermoElectron, Bremen, Germany). One third of the peptides in eachfraction were injected into a Zorbax peptide trap column(Agilent, Santa Clara, CA, USA) via the auto-sampler of theShimadzu UFLC so that they were concentrated and desaltedsimultaneously. The peptides were separated in a capillarycolumn (200 μm×10 cm) packed with C18 AQ (5 μm, 300 Å,Michrom BioResources, Auburn, CA, USA). The flow rate wasmaintained at 500 nl/min. Mobile phase A (0.1% FA in H2O) andmobile phase B (0.1% FA in ACN) were used to establish the60 min gradient comprising 45 min of 8–35% B, 8 min of 35–50%B and 2 min of 80% B followed by re-equilibrating at 5% B for5 min. The peptides were then analyzed on LTQ-FT with anADVANCE™ CaptiveSpray™ Source (Michrom BioResources) atan electrospray potential of 1.5 kV. A gas flow of 2, ion transfertube temperature of 180 °C and collision gas pressure of0.85 mTorr were used. The LTQ-FT was set to perform dataacquisition in the positive ion mode as previously describedexcept that an m/z range of 350–1600 was used in the full MSscan. Peptidemasseswere searchedNCBInr 20080822 (6,928,460sequences; 2,392,894,162 residues) and database search wasperformed using an in-house Mascot server (version 2.2.04,Matrix Science, Boston,MA,USA)with precursormass toleranceof 10 ppm andMS/MS tolerance of 0.8 Da. Twomissed cleavagesites of trypsin were allowed. Carbamidomethylation (C) was

set as a fixed modification, and oxidation (M), phosphorylation(S, T and Y) and deamidation (N) were set as variablemodifications. The obtained peptide/protein list for eachfraction was exported to Microsoft Excel.

2.9. Enzyme assays

The culture filtrate was harvested and concentrated bylyophilization (Freeze Dryer, FD-10M, Labfreez InstrumentsCo., Ltd, Beijing, P.R. China). All enzymatic assays were carriedout in sodium acetate buffer (100 mM, pH 5) at 30 °C in aBeckman Coulter DU 50 spectrophotometer (Brea, CA, USA).All assays were performed in triplicate and the mean valuescalculated. Carboxymethyl cellulase (CMCase), avicelase andxylanase were assayed by the modified method of Dinis et al.[26]. The reaction mixture for CMCase, avicelase or xylanaseassays contained 1 ml of crude enzyme solution and 1 mlof 1% of their respective substrates, namely carboxy methylcellulose (CMC), avicel or brichwood xylan (Sigma-Aldrich).After incubation at 30 °C for 30 min, the reducing sugars wereassayed by the dinitrosalicylic acid method [27]. One unit (U)of enzyme activity was defined as the amount of enzymereleasing 1 μmol of reducing sugar per minute.

TheMnP activitywasdetermined bymeasuring the oxidationof 2,6-dimethoxyphenol (DMP) at 469 nm (ε=49.6 mM−1 cm−1)at 30 °Cbyamodifiedmethodasdescribedpreviously [11]. Assaymixtures (1 ml) contained sodium acetate buffer (100 mM,pH 5.0), DMP (20 mM), MnSO4 (5 mM), H2O2 (10 mM) and 100 μlof an MnP sample (crude enzyme obtained from the spentsubstrate). One unit of MnP activity was defined as 1 μmolreaction product formed per minute.

Laccase activitywas determined by the oxidation of 2,2-azinobis (3-ethylbenzylthiozoline-6-sulfonate) (ABTS (1 mM)) as de-scribed previously [28]. One activity unit (U) is defined as theamount of laccase transforming 1 μmol/min ABTS to its cationradical (ε436 nm=29.3 mM−1 cm−1) in sodium acetate buffer(100 mM, pH 5) at 30 °C estimated by measuring the OD at436 nm. The assay mixture contained 100 μl enzyme (crudeenzyme obtained from the spent substrate), 100 μl of ABTSsubstrate (final concentration 1 mM) and 800 μl of sodiumacetate buffer (100 mM, pH 5.0).

2.10. Plate assays for cellulase, hemicellulase, laccase andperoxidase

Concentrated enzymes were introduced into different wellsfor plate assays (10, 20, 30, 40 and 50 μl of crude enzymeobtained from the spent substrate) as described previously[6,29]. The assay plates contained 1.8% agar and 0.5% CMC asa substrate for cellulase and 1.8% agar and 0.5% brichwoodxylan as a substrate for xylanase and were incubated over-night at 30 °C. The plates were subsequently stained for20 min with 1% Congo red and then destained, first with 1 MNaCl and then further with 0.5% acetic acid, which wasimmediately rinsed off with cold water. The clear hydrolytictransparent zones were observed against the red backgroundas indicated for cellulase and xylanase activities, respectively.

For MnP, the assay plates contained 1.8% agar, 20 mMDMP,5 mM MnSO4 and 10 mM H2O2 as a substrate and wereincubated at 30 °C for overnight. For laccase, the assay plates

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contained 1.8% agar and 1 mM guaiacol as a substrate andwere incubated at 30 °C overnight. The clear oxidative darkbrown zones were observed against the white background asan indicator of laccase and peroxidase activity.

2.11. Zymography

The activities of the various enzymes were determined byzymography on native-PAGE as described previously [30,31].In brief, 10 μg of total protein obtained from sugarcane ba-gasse spent substrate was loaded onto native-PAGE for eachlignocellulolytic enzyme zymography. The gels were preparedwith 0.1% CMC and xylan for cellulase and hemicellulaseactivities, respectively. The native-PAGE was run in a MiniProtean (Bio-Rad Laboratories, Hercules, CA, USA) gel runningsystem for about 120–180 min at 100 V; the gels were washedwith sodium acetate buffer (100 mM, pH 5) containing 40%isopropanol. The enzyme reaction was performed at 30±2 °Cafter overnight renaturation at 4 °C and cellulase and xylanasebands were developed with 1% Congo red staining solution.

For the determination of the MnP activity, gels wereincubated in sodium acetate buffer (100 mM, pH 5.0) with20 mM DMP, 5 mM MnSO4 and 10 mM H2O2. The reaction wasstopped by immersing the gels in 50% (v/v)methanol containing10% (v/v) acetic acid at 50 °C. The gels were then immersed in20% trichloroacetic acid to darken the DMP oxidation product.Zymography for laccase activity was performed onnative-PAGEusing 1 mM guaiacol as substrate in sodium acetate buffer(100 mM, pH 5.0) as described previously [29].

3. Results

3.1. Cropping period, crop yield and quality traitsassessment

A high biological efficiency (BE) of 80±15% was obtained whenG. lucidumwas grownwith sugarcanebagasseas a lignocellulosesubstrate (Table 1) which was 2–2.6 fold better than previouslyreported by others [32,33].When totalG. lucidum productionwasanalyzed on the basis of the total dry weight per bag (150 g),we found that itwas significantlyhigher than theyield obtainedby others [34]. While we used sugarcane bagasse alone as alignocellulose substrate for the cultivation of G. lucidum, othersapplied a combination of sawdust, paddy straw and rice bran(22.5:67.5:10), sawdust and stillage grain (4:1) or sawdust and teawaste (80:20) as a lignocellulose substrates [32–34].

Table 1 – Cropping period, crop yield, quality traits assessmensugarcane bagasse as lignocellulose substrate.

Different stagesof mushroom

growth

Incubation(days)

Culture condi

Temperature(°C)

Light(photon m−2 s−

Mycelia growth 10–15 30 –Primordial initiation 15–20 30±2 4Stipe growth 20–30 28±2 6Pileus maturation 30–35 30±2 15 (12 h/day)Fruit body harvesting 40 30±2 15 (12 h/day)

3.2. Functional classification of the identified extracellularproteins

The secretome of G. lucidum was characterized when cultivat-ed in sugarcane bagasse as a lignocellulosic substrate. The71 identified proteins were classified functionally accordingto their biological role. Fig. 2 (see also Table 2) depicts thefunctional classification of the G. lucidum secretome whichwas sub-grouped into cellulases (24%), hemicellulases (5%),glycoside hydrolases (10%), lignin depolymerizing proteins(24%), a protease (2%), phosphatases (7%), transport proteins(10%) and hypothetical proteins (18%).

The distribution of the identified proteins with respect totheir molecular weights and isoelectric points is presented inFig. 3. The molecular weights of the identified proteins rangedfrom 20 to 120 kDa. Interestingly, the range of the isoelectricpoints was quite narrow (4.0–5.6) though a few proteins hadalso an isoelectric point in the range of 7.2–9.2 (Fig. 3).

3.3. Identification of proteins involved in cellulosehydrolysis

Cellulases comprise at least three major groups of enzymesthat are involved in the hydrolysis process of cellulose toglucose (endo 1,4-beta glucanase, exo 1,4-beta glucanase andbeta glucosidase) [26,35,36]. Accordingly, in our study weidentified several cellulose hydrolyzing enzymes (10), suchas endo 1,4-beta glucanase, that cuts 1,4-beta glucosidic bondsof cellulose fiber and creates free chain ends, exo 1,4-betaglucanase, that cuts the free chain ends of disaccharidecellobiose, and beta glucosidase, which hydrolyzes the cellobioseto glucose. Transaldolase catalyzes the reversible transfer of adihydroxyacetone moiety derived from D-fructose-6-phosphatetoD-erythrose-4-phosphate, forming sedoheptulose-7-phosphateand releasing glyceraldehyde-3-phosphate. The identificationand functional classification of G. lucidum-derived cellulolyticproteins,when cultivated in sugarcane bagasse as a lignocellulosesubstrate, are presented in Table 2.

3.4. Identification of proteins involved in hemicellulosehydrolysis and glycoside hydrolase

The complete hydrolysis of xylan requires endo 1,4-betaxylanases, exo 1,4-beta xylanases, beta xylosidase, betaL-arabinofuranosidases, beta d-glucuronidases, acetylxylanes-terases, galactosidase, mannanases, mannosidase, polysaccha-ride lyase, glycoside hydrolase and phytase [14,26,37,38]. In our

t and biological efficiency of G. lucidum when cultivated in

tion Number ofprimordialinitiation(per bag)

Fresh weightof fruit body

(g)

Biologicalefficiency

(%)1)RH(%)

CO2 conc.(%)

65–75 5 15±3 400±50 80±1590–95 0.1–0.570–80 0.1–185–95 0.04–0.0585–95 0.04–0.05

Fig. 2 – Pie chart depicting the identified proteins of G. lucidum when cultivated in sugarcane bagasse as a lignocellulosesubstrate. Representation of proteins, identified by LC-MS/MS and classified according to their function gene ontology (GO)category. In addition to the percentage (%), the absolute number of proteins is shown.

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investigation, we identified endo 1,4-beta xylanase and alphagalactosidase from the culture filtrate of G. lucidum. Amongthem, endo 1,4-beta xylanase is the most important enzymewhich splits the glycosidic bonds to produce short oligosaccha-rides of various lengths. The ability of alpha galactosidase is tocatalyze the removal of alpha-linked terminal non-reducinggalactose residues from small oligosaccharides as well as fromgalactopolysaccharides and galactolipids. Glycoside hydrolasescleave internal 1,4-beta xylosidic bonds in heteroxylans andderived xylo-oligosaccharides, producing unsubstituted orbranched xylo-oligosaccharides. In this study, we identified4 glycoside hydrolase enzymes from the culture filtrate ofG. lucidum. The identification and functional classification ofG. lucidum-derived hemicellulolytic proteins and glycoside hy-drolases, when cultivated in sugarcane bagasse as a lignocellu-lose substrate, are presented in Table 2.

3.5. Identification of proteins involved in lignindepolymerization and other proteins

Enzymes involved in lignin depolymerization comprise laccase,peroxidase, glucose oxidase, isoamyl alcohol oxidase, glutathi-one reductase, glutathione S-transferase, copper radical oxi-dase, cellobiose dehydrogenase (CDH) and others [26,30,39].Here,we identified several such lignindepolymerizing enzymesincluding five laccases, a MnP, a cellobiose dehydrogenase, anacyl-CoA dehydrogenase and two glutathione reductase iso-enzymes. Laccase is known to oxidize phenolic lignin struc-tures. Peroxidases catalyze the H2O2-dependent oxidativedegradation of lignin and other similar compounds. CDH is anextracellular hemoflavoprotein, with separate heme and flavindomains. It also often contains a carbohydrate binding module(CBM) [40–42].

Several other proteins, including a protease, four transportproteins, three phosphatases and several hypothetical pro-teins were identified from G. lucidum cultivated in sugarcanebagasse as a lignocellulosic substrate (Table 2).

3.6. Enzyme assay, plate assay and zymography

Fig. 4A presents the zymogram analyses of cellulase, xylanaseand the two lignin depolymerizing enzymes laccase andMnP. In Fig. 4A(1) three intense bands of cellulases wereobserved in the zymogram-PAGE amended with 0.1% of CMCas substrate thus suggesting a diversity of G. lucidum cellulases(Fig. 4A(1)). Similarly, one intense band of a hemicellulaseenzyme was observed when the zymogram was amendedwith 0.1% of brichwood xylan as substrate (Fig. 4A(2)). Thelignin depolymerizing enzyme MnP was observed when thezymogram was stained with DMP (20 mM), MnSO4 (5 mM)and H2O2 (10 mM) as substrate (Fig. 4A(3)). Similarly, in thelaccase zymogram four laccase isoforms were found whenthe zymogram was stained with ABTS (1 mM) as substrate(Fig. 4A(4)). Fig. 4B depicts the hydrolysis zones of cellulose (1)(6–10 mm) and xylan (2) (6–8 mm) caused by cellulase andxylanase activities, respectively. Similarly, the oxidative zonesfor the peroxidase activity (3) (6–9 mm) and laccase activity (4)(6–11 mm) were observed.

The time-dependentproductionof thevarious lignocellulolyticenzymes (endo 1,4-beta glucanase, exo 1,4-beta glucanase, endo1,4-beta xylanase, laccase and MnP-peroxidase) was studiedbetween 5 and50 days in G. lucidum and presented in Fig. 5.Among the different lignocellulolytic enzymes laccase and MnPare the major oxidative enzymes for the degradation of ligninand reached an earlier peak on day 15 for laccase (55 U/g) and onday 20 forMnP (42 U/g),while additional delayedpeaks for laccase(83 and 93 U/g) and MnP (75 and 86 U/g) were observed on days30 and 40, respectively. This variability of enzyme productionssuggests that e.g. more than one laccase isoenzyme was secreted(compare with Fig. 4) and that the timed-dependent G. lucidumenzyme secretome depends on the culture conditions [43].Similarly, a double-peak laccase secretion (on days 5 and 14(30 and 100 U/kg), respectively) has been described for Ganodermaaustralewhen cultivated inwood chips ofD.winteri (Drimyswinteri)[43]. However, a single-peak laccase and MnP secretion for

Table 2 – Identification and functional classification of lignocellulose degrading proteins in G. lucidum when cultivated insugarcane bagasse as a lignocellulose substrate.

Protein name a NCBI ID Molecularmass

pI Score PredictedSignalP b

Total no. ofpeptide matches

Cellulolytic enzymesExoglucanase 1 precursor gi|121852 56,077 5.21 270 Y 19Cellulase gi|453224 55,070 4.68 281 Y 13Exocellobiohydrolase gi|913560 48,984 5.04 215 Y 16Endoglucanase gi|55295400 42,673 4.67 129 Y 9Cellobiohydrolase I gi|165879995 19,164 3.91 113 N 3Cellulose 1,4-beta-cellobiosidase precursor gi|146350520 50,287 5.27 65 Y 7Exo-beta-1,3-glucanase gi|73808014 81,656 4.74 57 Y 1Glucan 1,3-beta-glucosidase gi|145255120 45,782 4.96 82 Y 3Exo cellobiohydrolase gi|169852726 49,745 5.56 59 Y 1Transaldolase, putative gi|146082650 37,234 4.55 145 N 11

Hemicellulolytic enzymesEndo-1,4-B-xylanase B gi|12006977 30,805 5.43 84 Y 2Alpha-galactosidase gi|10944324 48,997 4.59 69 Y 13

Glycoside hydrolasesGlycoside hydrolase family 74 gi|162138864 88,665 5.02 200 N 7Glycoside hydrolase family 35 protein gi|170111653 120,129 5.66 109 N 9Endo-1, 4-beta mannanase gi|110627663 49,126 4.24 58 Y 1Endo-1, 4-beta mannanase gi|110627661 49,369 4.83 122 Y 2

Lignin-degrading enzymesManganese peroxidase gi|6580077 38,678 4.19 112 Y 14Cellobiose dehydrogenase gi|1279638 82,092 5.06 267 Y 8Laccase gi|38490397 32,184 4.79 170 N 8Laccase 2 gi|37813119 43,016 4.56 110 N 151Laccase LCC3-1 gi|9957143 56,043 4.71 107 Y 5Laccase gi|146327854 42,332 4.56 95 N 31Laccase 2 gi|21616728 57,178 4.53 61 Y 1Acyl-CoA dehydrogenase-like protein gi|108804881 44,481 5.52 55 N 4Glutathione reductase gi|1150524 49,800 8.73 67 N 4Glutathione reductase gi|22537522 48,910 5.30 67 N 4

Protease enzymeProtease gi|6981420 26,627 4.71 71 Y 104

Phosphatase enzymesAcid phosphatase gi|58265204 60,001 4.50 62 Y 5Diguanylate cyclase/phosphodiesterase gi|154248006 79,400 5.45 55 N 2Histidinol-phosphate aminotransferase gi|121701171 48,354 5.12 55 N 2

Hypothetical proteinsHypothetical protein gi|114667194 127,093 4.81 179 N 14Hypothetical protein BC1G_03567 gi|154317330 109,929 5.42 132 Y 20Hypothetical protein GFO_1847 gi|120436198 30,419 4.96 71 N 7Hypothetical protein BC0263 gi|30018500 38,513 9.22 61 Y 4Hypothetical protein CC1G_12248 gi|169865399 78,636 4.95 59 N 5Hypothetical protein PGUG_04883 gi|146413915 49,602 4.66 57 Y 9Hypothetical protein LOC495267 gi|147900165 63,163 8.14 56 N 3Hypothetical protein bll3994 gi|27379105 21,868 9.92 55 N 4

Transport proteinsOligopeptide ABC transporter gi|52079641 60,996 5.54 70 Y 1Lipid transfer protein gi|10803445 10,281 8.89 64 Y 5Biopolymer transport ExbD/TolR family protein gi|158336546 14,597 5.10 57 Y 6Phosphate transporter subunit gi|157368261 36,634 8.89 155 N 5

a Assignment of proteins to those in the NCBInr database (www.ncbi.nlm.nih.gov/) was determined by LC-MS/MS of protein bands obtainedfrom the SDS-PAGE gel. Names or predicted functions of conserved domains in the identified proteins were retrieved from the NCBInr database(www.ncbi.nlm.nih.gov/) to which significant peptide matches (P<0.05) were made using the Mascot search engine.b Signal peptide prediction by SignalP (www.cbs.dtu.dk/services/SignalP/): Y, yes; N, no.

304 J O U R N A L O F P R O T E O M I C S 7 7 ( 2 0 1 2 ) 2 9 8 – 3 0 9

Fig. 3 – Simulated 2D gel presentation of the secretomeobtained from G. lucidum cultivated in sugarcane bagasse.The proteins identified by LC-MS/MS were uploaded ontoJVirGel (http://www.jvirgel.de/), an online software used togenerate a virtual 2D gel image; molecular weight (MW) andisoelectric point (pI).

Fig. 4 – Zymogram and plate assay of ligninocellulolyticenzymes (secretome proteins) from G. lucidum. (A) Fourzymograms of the secretome proteins from G. lucidum.Lane 1: zymogram of cellulase with three isoforms (arrows)when the gel was incubatedwith 0.1% of CMC and stainedwith1% Congo red, Lane 2: zymogram of xylanase (arrow) when thegel was incubated with 0.1% of brich wood xylan as substrateand stained with 1% Congo red, Lane 3: zymogram ofMnP/peroxidase (arrow) when the gel was stained with 20 mMDMP, 5 mMMnSO4 and 10 mMH2O2, Lane 4: zymogram of

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G. applanatum (Ganoderma applanatum) on day 28 (216 and 271 U/l)has been observed when cultivated in wheat straw as substrate[26], thus pointing at the culture conditions that influence thesecretome crucially.

Hydrolytic enzymes, namely endo 1,4-beta glucanase, exo1,4-beta glucanase and endo 1,4-beta xylanase, were quanti-fied and presented in Fig. 5 too. Exo 1,4-beta glucanasereached a double-peak maximum production on days 15 and40 (34 and 48 U/g) while endo 1,4-beta glucanase reached amaximum production on day 20 (26 U/g) only though it wasslightly increased during days 40–45. Similarly, a double-peakendo 1,4-beta glucanase secretion (on days 21 and 42 with200 U/kg) has been described for G. australe when culturedwith wood chips of D. winteri [43]. In contrast, a single-peakexo 1,4-beta glucanase and endo 1,4-beta glucanase produc-tion on day 28 (3 and 67 U/l, respectively) could be observedfor G. applanatum when cultivated in wheat straw [26].

`Endo 1,4-beta xylanase is the major hydrolytic enzyme forthe degradation of hemicellulose and it reached a maximumdouble-peak enzyme production on days 25 and 40 (20 and33 U/g). In contrast, the same enzyme reached only a single-peak maximum production after 5 days (800–1000 U/kg) forG. australewhen culturedwithwood chips of D. winteri [43] anda single-peak maximum production after 28 days (132 U/l) forG. applanatumwhen cultivated inwheat strawas substrate [26].

laccase (arrows) with four isoforms when the gel was stainedwith 1 mM guaiacol as substrate. (B) Different lignocellulolyticenzyme activities qualitatively assayed in petri dishes.B1, B2, B3 and B4: lignocellulolytic plate assays of cellulase,hemicellulase/xylanase, MnP and laccase, respectively(c, control (without enzyme); i, 10 μl; ii, 20 μl; iii, 30 μl; iv, 40 μl;v, 50 μl of crude enzyme obtained from spent substrates ofsugarcane bagasse). The assay plate for cellulase (B1) andhemicellulase (B2)was stainedwithCongo red (1%), forMnP (B3)was stained with DMP, MnSO4 and H2O2 (20 mM, 5 mM and10 mM) and for laccase (B4) was stained with guaiacol (1 mM).

4. Discussion

Due to the shortage of fossil fuel and its impact on globalwarming there is an urgent need to produce and use alter-native renewable energy resources such as biofuel [3,4,44].Sugarcane bagasse is an agricultural residue and generallyused as a fuel that consists of various polysaccharides such ascellulose (50%), hemicellulose (25%) and lignin (25%) [10].Hence, for the future biofuel generation there is a requirement

Fig. 5 – Ligninolytic enzyme activity of G. lucidum grown insugarcane bagasse as a lignocellulose substrate. Enzymeswere harvested every 5 days up to 50 days and enzymeactivities were analyzed as describe in Materials andmethods. Values represent data obtained in triplicate fromtwo independent experiments (* P<0.05, compared withcontrols).

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for the development of a hydrolysis method of this biopoly-mer into a fermentable sugar. In the present study, we reportfor the first time the G. lucidum secretome analysis that iscultured with the lignocellulosic biomass of sugarcane ba-gasse. In total, 71 secreted proteins were identified, of which24% are cellulases, 5% are hemicellulases and 24% are lignindepolymerizing proteins.

Till today, cellulolytic enzymes of G. lucidumwere character-ized by a few researchers only [12,14] and thus our comprehen-sive study demonstrates for the first time theG. lucidum‐derivedsecretome and its lignocellulolytic proteins.

4.1. Cellulolytic enzymes

The effective degradation of lignocellulosic biomass dependsupon the appropriate levels of endo 1,4-beta glucanase, exo1,4-beta glucanase and beta glucosidase secretion [35]. Table 2shows the expression of 10 cellulose hydrolyzing proteins.Though a few reports have reported about G. lucidum-derivedcellulases [12,14], our analysis reveals for the first time theidentification of: 7 exo 1,4-beta glucanase, 1 endo 1,4-betaglucanase, 1 beta glucosidase and1 transaldolase fromG. lucidumwhen cultivated in sugarcane bagasse as lignocellulosic sub-strate. In addition to G. lucidum, several other fungi, such asTrichoderma reesei, Aspergillus niger, Phanerochaete chrysosporiumand Penicillium janthinellum, produce cellulase and hydrolyzecellulose [30,45,46]. However, though T. reesei has the potentialto produces exoglucanases, endoglucanases and beta glucosi-dase that are essential for cellulose degradation, the cellulosehydrolysis efficiency is limited due to a lower content of betaglucosidase [46]. Similarly, P. chrysosporium has the potential toproduce lignocellulolytic enzymes, but the major disadvantageis the instability of its enzyme production [47]. Other importantcellulase-producing fungi are Penicillium and Aspergillus, whichare able to secrete large amounts of beta glucosidase. However,the total cellulase activity (FPase) found in their enzymaticextracts is relatively low [48]. Further, the major advantage of

G. lucidum is that it can produce all the three major cellulolyticenzymes such as exoglucanases, endoglucanases, and betaglucosidase [12]. The cellulolytic zymography also revealed thehydrolyzing potential of G. lucidum.

4.2. Hemicellulolytic and glycoside hydrolase enzymes

Hemicelluloses are composed of pentoses and hexoses andtherefore, the second most abundant renewable biomassaccounting for 25% of the lignocellulosic biomass [10,49]and also an important source of fermentable sugars forbiorefining applications. Hemicelluloses from hardwood con-tain mainly xylans while in hemicelluloses from softwoodglucomannanases are most abundant [35]. Here, in additionto cellulases, the major hemicellulolytic enzymes (endo 1,4-beta xylanase and alpha galactosidase) were also identifiedfrom G. lucidum when cultivated in sugarcane bagasse as alignocellulosic substrate and hence these enzymes have thepotential to convert complex hemicellulose into simple sugarsor oligosaccharides [35,50]. The endo 1,4-beta xylanase andalpha galactosidase enzymes were previously characterizedfrom G. lucidum [13,14]. Our hemicellulolytic zymography alsorevealed the hydrolyzing potential of G. lucidum. In addition,our study discloses four new glycoside hydrolases in theG. lucidum-derived secretome (such as glycoside hydrolasefamily 74, glycoside hydrolase family 35 protein and twoendo 1,4-beta mannanases). The glycoside hydrolase enzymehydrolyzes the glycosidic bond between a carbohydrate and anon-carbohydrate moiety. Though these glycoside hydrolaseenzymes have been identified from P. chrysosporium already[6], to the best of our knowledge, this is the first report forthe identification of these glycoside hydrolases at the proteinlevel from theG. lucidum-secretome. However, Liu et al. reportedvery recently 216 putative glycoside hydrolase genes fromG. lucidum [51,52].

4.3. Lignin-degrading enzymes

Lignin peroxidase (LiP), MnP and laccase were generallyconsidered as lignin degrading enzymes [11,15,39]. The ex-pression of MnP, laccases, cellobiose dehydrogenase, gluta-thione reductases and acyl-CoA-dehydrogenase was foundin the G. lucidum secretome when cultivated in sugarcanebagasse to degrade lignin through oxidases. Cellobiose dehy-drogenase is an extracellular redox enzyme of ping-pongtype that generates hydroxyl radicals by reducing Fe3+ toFe2+ and O2 to H2O2 and it plays an important role in bothlignin and cellulose hydrolysis by (i) breaking beta-ethers,(ii) demethoxylating aromatic structures in lignin and (iii)introducing hydroxyl groups in non-phenolic lignin [40,53].Furthermore, cellobiose dehydrogenase can be converted intoa lignin degrading cellulose-quinone oxidoreductase (CBQ)by proteolytic cleavage [54]. However, a detailed research onthe cellobiose dehydrogenase protein is required to tailorits exact role in lignin degradation and elucidate its lignindegradation-mediated mechanism; the information of thecellobiose dehydrogenase gene, identified in G. lucidum by Liuet al. might be also useful [51].

The LiP and MnP require H2O2 for their catalytic activitiesand the major sources of H2O2 are various lignin-related

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phenolic compounds after being oxidized by laccase [11,55]. Weidentified in the G. lucidum secretome five laccase isoforms,a MnP and two glutathione reductase isoenzymes (Table 2).Four laccase isoenzymes and one MnP were also confirmed byzymography and plate assays (Fig. 4), which differ from threeG. lucidum laccase isoforms identified by others [56]. Though wecould describe here only one G. lucidum-derived MnP isoform,it might be possible that more than only one isoform exists inG. lucidum because others have described several MnP isoformse.g. in Ganoderma sp. YK-505 [57]. A recent report from Liu et al.identified 16 laccase genes and 7 peroxidase genes fromG. lucidum [51]. Similarly, other basidiomycetes, suchas Pleurotussapidus, were also found to express MnP and versatile peroxi-dases when cultivated in peanut shell and glass wool, respec-tively [58]. Both laccase and MnP zymograms demonstratethe lignin degradation potential for these G. lucidum-derivedenzymes.

4.4. Other proteins

In addition to the lignocellulolytic proteins, one protease,four transport proteins, three phosphatases and severalhypothetical proteins were also identified when G. lucidumwas cultivated in sugarcane bagasse as a lignocellulose sub-strate. However, the biological significance of their produc-tion remains unclear. The production of a protease duringlignocellulose degradation has been reported, but it remainsuncertain whether extracellular peroxidases are substan-tially degraded under ligninolytic culture condition [59,60].Nevertheless, protease actions in cellulolytic cultures werecorrelated to the activation of the cellulase activity [61]and to the cleavage of cellobiose dehydrogenase functionaldomains [62]. A serine protease and metallo-protease havealso been reported from P. sapidus [58].

5. Conclusion

This study reports for the first time about a comprehensiveG. lucidum secretome analysis when cultivated in sugarcanebagasse as lignocellulose substrate. We could demonstratethe expression of lignocellulolytic enzymes, including severalcellulases (exo and endo glucanase, cellobiase), hemicellulases(endo xylanase, alpha galactosidase), a cellobiose dehydroge-nase, glycoside hydrolases, a transaldolase, an acyl-CoA-dehydrogenase, a MnP, laccases, glutathione reductases, phos-phatases and a protease. Hence, G. lucidum is a potentialcandidate for the production of lignocellulolytic enzymes andcapable of degrading a complex lignocellulose substrate intosimple sugars for the generation of future biofuel.

Acknowledgments

The authors thank the University Grants Commission forproviding the scholarship to T.M. through Science for Merito-rious Students. The authors also thank Dr. R. Rengasamy, CASin Botany, University of Madras, for providing the adequatelaboratory facility to carry out the research. Furthermore, thisstudy was supported by the Institute of Advanced Studies,

Nanyang Technological University (NTU), Singapore. Theauthors also thank Dr. S.K. Sze (School of Biological Sciences,NTU, Singapore) for technical assistance.

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