GENOMICS, TRANSCRIPTOMICS, PROTEOMICS

Proteomic analysis in non-denaturing conditionof the secretome reveals the presence of multienzymecomplexes in Penicillium purpurogenum

Alvaro Gonzalez-Vogel & Jaime Eyzaguirre &

Gabriela Oleas & Eduardo Callegari & Mario Navarrete

Received: 18 August 2010 /Revised: 5 October 2010 /Accepted: 12 October 2010 /Published online: 23 October 2010# Springer-Verlag 2010

Abstract Proteins secreted by filamentous fungi play keyroles in different aspects of their biology. The fungusPenicillium purpurogenum, used as a model organism, isable to degrade hemicelluloses and pectins by secreting avariety of enzymes to the culture medium. This work showsthat these enzymes interact with each other to form highmolecular weight, catalytically active complexes. By usinga proteomics approach, we were able to identify severalprotein complexes in the secretome of this fungus. Theexpression and assembly of these complexes depend on thecarbon source used and display molecular masses rangingfrom 300 to 700 kDa. These complexes are composed ofa variety of enzymes, including arabinofuranosidases,acetyl xylan esterases, feruloyl esterases, β-glucosidasesand xylanases. The protein–protein interactions in thesemultienzyme complexes were confirmed by coimmuno-precipitation assays. One of the complexes was purifiedfrom sugar beet pulp cultures and the subunits identified

by tandem mass spectrometry. A better understanding ofthe biological significance of these kinds of interactionswill help in the comprehension of the degradationmechanisms used by fungi and may be of special interestto the biotechnology industry.

Keywords Xylanosome .Penicillium purpurogenum .

Sugar beet pulp . Hemicellulose degradation .Multienzymecomplexes . Secretome

Introduction

Cellulolytic and hemicellulolytic enzyme systems from anumber of microorganisms have been studied due to theirpotential in generating products of industrial interestderived from the hydrolysis of lignocellulosic material(Lee 1997; Saha 2003; Tabka et al. 2006) or for otherbiotechnological applications (Benoit et al. 2007; Sigoillotet al. 2005). Lignocellulose is composed mainly ofpolysaccharides (hemicelluloses, pectins and cellulose)and lignin, which are associated in plant cell walls(Cosgrove 2005). Hemicelluloses and pectins are abundantpolysaccharides present in agroindustrial residues andtheir degradation represents a challenge since the individ-ual components show different degrees of resistance tohydrolysis. In addition, the composition and solubility ofthese materials vary among the species and plant tissues(Saha 2003).

Hemicelluloses are heterogeneous polysaccharides andcorrespond to the second most abundant organic structurein plant cell walls (Polizeli et al. 2005). They represent acomplex mixture in which the main component is xylan.This compound is a linear chain of D-xylopyranoses β-1,4linked and it may be substituted by L-arabinose and acetic,

Electronic supplementary material The online version of this article(doi:10.1007/s00253-010-2953-0) contains supplementary material,which is available to authorized users.

A. Gonzalez-VogelDepartamento de Genética Molecular y Microbiología,Pontificia Universidad Católica de Chile,Alameda 340,Santiago, Chile

J. Eyzaguirre :G. Oleas :M. Navarrete (*)Facultad de Ciencias Biológicas, Universidad Andrés Bello,República 252,Santiago, Chilee-mail: mar.navarrete@uandresbello.edu

E. CallegariDivision of Basic Biomedical Sciences,Sanford School of Medicine, University of South Dakota,Vermillion, SD, USA

Appl Microbiol Biotechnol (2011) 89:145–155DOI 10.1007/s00253-010-2953-0

hydroxycinnamic and glucuronic acids (de Vries and Visser2001). Pectins are complex polysaccharides of heteroge-neous composition, constituted by “hairy” and “smooth”regions. The smooth regions (homogalacturonic pectins) arelinear chains of D-polygalacturonic α-1,4 linked and arepartially methylated and acetylated. Smooth regions can beinterrupted by hairy regions (rhamnogalacturonic pectins),mainly composed of rhamnogalacturonans branched witharabinans or arabinogalactans (Saulnier and Thibault 1999).

In nature, there are numerous microorganisms, mainlybacteria and filamentous fungi that synthesize a widevariety of plant cell-wall degrading enzymes. Theseorganisms can thus use lignocellulose polysaccharides asnutrients and incorporate the hydrolysis products into theirmetabolism (Williamson et al. 1998). The biodegradation ofthese polysaccharides is an elaborate process where severaldifferent enzymes act in a coordinated fashion over both themain chain and the ramifications of hemicelluloses andpectins. Xylanolytic enzymes such as 1,4-β-xylanases (EC3.2.1.8) and β-xylosidases (βXYL, EC 3.2.1.37) break themain chain of xylan; pectate lyases (EC 4.2.2.10) andgalacturonidases (EC 3.2.1.67) cut the main chain ofpectins; α-L-arabinofuranosidases (ABF, EC 3.2.1.55),acetyl xylan esterases (AXE, EC 3.1.1.72), feruloylesterases (FAE, EC 3.1.1.73) and α-D-glucuronidases (EC3.2.1.1) are debranching enzymes that facilitate the degra-dation by the main chain acting enzymes for a completehydrolysis of xylan and pectins (Faulds et al. 2006). Theheterogeneous composition of the polysaccharides isresponsible for the production of multiple enzymes withdifferent substrate specificities. By acting in a synergisticway a successful degradation is achieved (de Vries et al.2000). Synergy can best be accomplished through stableprotein–protein contact among enzymes thus generatingcomplex structures. These are called cellulosomes (havingactivity on cellulose) or xylanosomes (acting on xylan).These complex structures have been identified in somebacteria and a thermophilic fungus (Gal et al. 1997; Goldand Martin 2007; Ohtsuki et al. 2005). However, onlycellulosomes have been confirmed at a genetic level (Bayeret al. 1998). Xylanosomes have been shown to have animportant role in the degradation of hemicelluloses (Jiang etal. 2005; Lee et al. 1993; Pason et al. 2006). This kind oforganization is needed to minimize diffusion and loss ofproducts and intermediates (Waeonukul et al. 2008),increasing substrate availability (Karboune et al. 2008),and to generate synergism and stability (de Vries et al.2000; Mukhopadhyay et al. 2003). Little is known aboutthe composition, functional properties and assembly of thexylanosomes (Jiang et al. 2006; Waeonukul et al. 2008).

Among lignocellulolytic microorganisms, fungi haveshown a high ability to secrete a wide range of xylanases;Aspergillus, Trichoderma and Penicillium are the most

studied fungi (Chávez et al. 2006). In our laboratory, wehave used as a model organism Penicillium purpurogenum,a soft-rot fungus isolated from Chilean forest soil (Musalemet al. 1984), which is an active enzyme producer whengrown on different carbon sources (Steiner et al. 1994).Some of these proteins have been purified and characterized.Such is the case for two endoxylanases (Chávez et al. 2002),β-glucosidase (Hidalgo et al. 1992), two acetyl xylanesterases (AXE I and AXE II) (Egaña et al. 1996; Gordilloet al. 2006), and three arabinofuranosidases (de Ioannes et al.2000; Fritz et al. 2008; Ravanal et al. 2010).

In this work, we use a proteomics approach, a newimmunological strategy and biochemical techniques inorder to study protein–protein interactions and to isolatecatalytically active protein complexes. Besides, we intendto demonstrate that these proteins do interact and that thecomposition of the complexes is different depending on thecarbon source.

Materials and methods

Fungal strain, culture media and enzyme source

P. purpurogenum ATCC Nº MYA-38 was kept on 2.4%potato dextrose agar plates (Difco) and grown at 28 °C.Spores were inoculated and grown in Mandel’s liquidmedium pH 5.0 (Mandels and Weber 1969) as describedpreviously (Hidalgo et al. 1992). Liquid cultures were usedfor enzyme production with the indicated carbon sources at1% (sugar beet pulp, corn cob or acetylated xylan) in arotary shaker (150 rpm) at 28 °C, and a mixture of urea(0.3 gL−1), ammonium sulphate (1.4 gL−1), and neopeptone(0.75 gL−1) as nitrogen source. After 6 days of growth, thecultures were filtered, centrifuged (30 min, 20,000×g at 4 °C) and the supernatant was concentrated by ultrafiltrationusing a Pellicon PTGC membrane (Millipore Corp.) with a10,000 Da cutoff.

Isolation of complexes

The concentrated supernatant was fractionated with ammo-nium sulphate (40–90%). To separate the complexed fromthe noncomplexed enzymes the precipitate was resuspendedin 50 mM phosphate buffer pH 6.0 containing 150 mMNaCl at 4 °C and subjected to tandem gel filtration in0.75×90 cm columns of Sephacryl HR S200 coupled toSephacryl HR S300 (Sigma, St. Louis, MO, USA) usingthe same buffer at a flow rate of 24.6 mLh−1. The elution ofthe standard proteins immunoglobulin M (900 kDa), ferritin(440 kDa), bovine serum albumin (66 kDa), cytochrome C(12.4 kDa) and the polysaccharide Dextran Blue(>1,500 kDa) was conducted in the same manner.

146 Appl Microbiol Biotechnol (2011) 89:145–155

Purification of a complex

A sugar beet pulp culture supernatant was precipitated with90% ammonium sulphate and then was resuspended in50 mM citrate buffer pH 4.0, and loaded in a BlueSepharose column 80 cm long. Two proteins peaks weredetected at 280 nm, one in the wash and the other in asaline gradient from 0 to 1 M NaCl. The protein peak in thegradient was active when assayed with p-nitrophenylacetate and the other peak was inactive. Active fractionswere pooled, dialyzed with 50 mM acetate buffer pH 5.0overnight and loaded in a carboxymethyl cellulose 50column 30 cm long. An active protein peak was obtained in thewash. This peak was further purified in a hydroxylapatitecolumn 5 cm long. Proteinswere eluted with 10mMphosphatebuffer pH 6.0. One active peak was detected and the purity ofthe complexes was analyzed by Blue Native PAGE.

Identification of bands by mass spectrometry

The identification by mass spectrometry (MS) was per-formed according to the protocol described previously (Moet al. 2006). The eluted ions were analyzed by one fullprecursor MS scan (400–1,500 m/z) followed by fourMS/MSscans of the most abundant ions detected in the precursor MSscan while operating under dynamic exclusion or direct dataacquisition system (DDAS; see Electronic supplementarymaterial 1). Spectra obtained in the positive ion mode withnano-ESI-Q-Tof micro mass spectrometer (Micromass,UK)were deconvoluted, and analyzed using the MassLynxsoftware 4.1 (Micromass, UK). A peak list (PKL format)was generated to identify +1 or multiple charged precursorions from the mass spectrometry data file (processingparameters are detailed in Electronic supplementary Table 1).Mascot server v2.2 (www.matrix-science.com, UK) in MS/MS ion search mode (local license) was applied to conductpeptide matches (peptide masses and sequence tags) andprotein searches against NCBInr v20091218 (10227800sequences; 3491169761 residues) using the taxonomy filterfor fungi (645645 sequences). The parameters were set asdescribed previously (Mo et al. 2006). The protein redun-dancy that appeared at the database under different gi andaccession numbers were limited to fungi with the firstpriority assigned to Penicillium and the second priorityassigned to Aspergillus. All of the proteins identified in thecurrent study were found in these domains.

Bidimensional Blue Native gel coupledto zymogram—PAGE

Blue Native (BN) was performed in duplicate according toa modification of the protocol described previously (Wittiget al. 2006). All buffers were adjusted to pH 7.0 at 25 °C. A

3–12% native (without SDS) polyacrylamide gradient gelwith a 2.5% stacker was poured in a Bio-Rad Mini ProteanIII Cell using 1.0 mm spacers. The cathode buffer (15 mMBisTris/HCl, 50 mM Tricine pH 7) containing 0.02% (w/v)Coomassie Brilliant Blue G-250 and the anode buffer(50 mM BisTris/HCl pH 7) were chilled to 4 °C beforesamples (150 μg of protein) were loaded. 5 μL of nativemolecular weight standards (NativeMark, Invitrogen,Carlsbad, CA, USA) were used. Electrophoresis wasbegun at 40 V and 4 °C. After 1 h, the cathode bufferwas replaced by the same buffer containing 0.005% (w/v)of the dye, and the electrophoresis was continued at 60 V.After the dye front had run off the gels, one of the BN gelswas fixed (40% methanol and 10% acetic acid) and stainedwith colloidal Coomassie G250 (Candiano et al. 2004).The other gel was incubated in 50 mM phosphate bufferpH 6.0 for 5 min, and then incubated in the same buffercontaining 10 mgmL−1 methylumbelliferyl acetate (MUA,Sigma) or methylumbelliferyl dihydroferulate (dMUF)(Leschot et al. 2002) for 2 min and photographed underUV light. The lanes with acetyl or feruloyl esteraseactivity were cut out and washed with dissociatingsolution (1% SDS, 1% 2-mercaptoethanol) for 15 min atroom temperature. For a second dimension a 12% SDS gelwas poured in a Bio-RadMini Protean III Cell using a 1.5 mmspacer. The excised lanes were then inserted on top ofthe gel between the glass plate assemblies and sealedwith hot agarose solution comprising 1% agarose, 0.5%SDS, 15 mM 2-mercaptoethanol and 0.1% BromophenolBlue. Molecular weight standards (Bio-Rad, Hercules,CA, USA) were loaded in a separate lane. Electrophore-sis was performed at 80 V until the front reach thebottom of the gel. The gels were fixed and stained withSYPRO Ruby following the instructions of the manufac-turer (Molecular Probes, Invitrogen).

Enzymatic assays and protein determination

Enzyme activities were determined as follows for both theculture supernatants and the complexes. Acetyl esteraseactivity was assayed using p-nitrophenyl acetate (Sigma) assubstrate in 100-mM phosphate buffer at pH 6.0 and 37°Cas described before (Egaña et al. 1996). Product formationwas detected spectrophotometrically at 405 nm (Hewlett-Packard HP 8452A). 1 unit of activity was defined asthe amount of enzyme that liberates 1 μm p-nitrophenolper min.

Protein concentration in the chromatography eluates wasmeasured by absorbance at 280 nm. Otherwise, proteincontent was determined by the method of Bradford(Bradford 1976) using the Bio-Rad protein assay withbovine serum albumin as the standard according to themanufacturer’s instructions.

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Preparation of protein G Sepharose beadsfor coimmunoprecipitation

Protein G beads (5 mL; GE Healthcare, Piscataway, NJ,USA) were added to 45 ml of coimmunoprecipitation(coIP) buffer (CoB; 25 mM Tris HCl, pH 8.0, 150 mMNaCl, 0.5% deoxycholate, 0.2% SDS, and 1% Triton X-100) (Yamin et al. 1996), and incubated overnight at 4°Cwith constant agitation. The beads were centrifuged at6,000 × g for 10 min and washed three times with CoB.Thereafter, the beads were incubated in PBS buffer with 1%BSA (blocking buffer) for 60 min at 4°C, with constantagitation to diminish non-specific binding. Blocking bufferwas removed by centrifuging at 6,000×g for 5 min andwashing the beads four times with CoB. 0.05% sodiumazide was added in the fourth wash and the beads werestored at 4°C.

Fifty 50 microlitre Protein G Sepharose beads and5 μL of polyclonal antisera were added to 1.5 mlcentrifuge tubes containing 50 μg of a multienzymecomplex sample and vortexed for 20 s. Controls wereprocessed at the same time (beads and antibody). Thetubes were incubated overnight at 4°C with constantagitation. The antibody protein-bead suspension andcontrols were centrifuged at 20,000×g (30 min at 4°C).The pellet was washed three times with 1 ml of CoB andfinally with one volume of a low-salt buffer (25 mM TrisHCl, pH 8.0, 10 mM NaCl, 0.5% deoxycholate, 0.2%SDS, and 1% Triton X-100). To examine the resultingprotein complex, the pellet was mixed with 25 μL ofloading buffer (Laemmli 1970) 2X and boiled for 5 min todissociate the proteins. The samples were centrifuged at2,000×g for 15 min, and the supernatants were electro-phoresed on 12% sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and analyzed by Westernblots.

Antisera preparation, dot blots and Western blots

Antisera were prepared in rabbits as described previously(Belancic et al. 1995; de Ioannes et al. 2000; Egaña et al.1996) to produce polyclonal antibodies that recognizearabinofuranosidase 1 (ABF 1), arabinofuranosidase 3(ABF 3), acetyl xylan esterase I, (AXE I), acetyl xylanesterase II (AXE II) and xylanase A from P. purpurogenum.For dot blot analysis the coimmunoprecipitated sampleswere denatured with 14.4 mM 2-mercaptoethanol, 1% SDSand boiled. The native and denatured fractions (2 μl) wereplaced on a nitrocellulose membrane (Bollag and Edelstein1991). For Western blots the coimmunoprecipitated frac-tions were electrophoresed in an SDS-12% polyacrylamidegel (Laemmli 1970) and electro-blotted (or blotted) onto anitrocellulose membrane at room temperature for 1 h

(Bollag and Edelstein 1991). The immunoblot membraneswere incubated in blocking solution (TS buffer (50 mMTris, 150 mM NaCl) and 1.5% (v/v) milk (pH 7.5))overnight at 4°C. The membranes were then washed threetimes with TS buffer and treated with blocking solutioncontaining the corresponding primary antibody in a1:15,000 (v/v) dilution for anti-AXE1, a 1:2,500 dilutionfor anti-ABF1 and anti-ABF 3 and a 1:2,000 dilution foranti-XYLA and anti-AXE2. The incubation was performedat room temperature for 1 h. The membranes were washedagain three times with TS buffer and incubated with goatanti-rabbit immunoglobulin G conjugated with alkalinephosphatase (Sigma) in order to detect the antigen-antibodycomplexes (Towbin et al. 1979).

Analysis of arabinoxylan hydrolysate

For enzymatic hydrolysis of arabinoxylan (AX), thereaction mixture consisted of 2 μg of AX in 200 μL of50 mM potassium phosphate buffer pH 6.0, mixed with1 μg of purified complexes. It was incubated at 20°Cfor 24 h. One microlitre of the solution was spotted onTLC silica gel plates 37360 (Riedel-deHaën, Seelze,Gemany). The plates were developed with a mixture ofethyl acetate, acetic acid, distilled water (3:2:1) fol-lowed by heating for 5 min at 150 °C in an oven afterspraying the plates whit a solution containing 0.2%orcinol and 10% sulphuric acid in ethanol. One percentsolutions of arabinose, xylose, xylobiose, xylotriose,xylotetraose, xylopentaose, xylohexaose (Megazyme,Wicklow, Ireland) in 50 mM phosphate buffer pH 6.0were used as standards.

Results

Effect of the concentration and the precipitationon the formation of protein complexes

The first evidence for the presence protein complexes in P.purpurogenum culture supernatants was the protein patternand migration in the upper zone of a native electrophoresisin a polyacrylamide gradient gel (Fig. 1).

We analyzed the acetyl esterase activity by zymogramsof supernatants submitted to ammonium sulphate precipi-tation and concentration by ultrafiltration and compared thepattern with supernatants without treatment. The pattern issimilar, but the precipitated supernatants showed moreintense activity due to increased protein load. This ruled outthat the formation of complexes is a consequence of highsample concentration (Fig. 1). From here on, all theexperiments were performed with samples concentrated bysalt precipitation.

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Detection of complexes by 2D BN/SDS-PAGE

BN electrophoresis was performed to discard artifactualelements in the native gels. This technique allows proteinsto migrate exclusively based on molecular weight, sinceCoomassie Blue adds negative charges without breakingprotein–protein interactions. Figure 2 shows a separation ofproteins by BN gel electrophoresis of crude extracts ofcultures grown on different carbon sources. Since theproteins are not denatured it is possible to assay for enzymeactivities in the BN gel coupled to zymograms. Wescreened for acetyl esterase activity, observing bandsranging from 20 to 700 kDa with catalytic activity. The

fluorescence signal detected corresponds to a positivereaction with MUA (see “Materials and methods”). Aglucose-grown culture supernatant was used as a negativecontrol since this sugar is a catabolic repressor of thehemicellulolytic system (Chávez et al. 2006) (lane A,Fig. 2(1 and 2)). Four active bands are detected inacetylated xylan supernatant; one of them is a lowmolecular weight protein, and the other three appear inbands of over 150 kDa (lane D, Fig. 2). Both sugar beetpulp and corn cob induced two complexes (one of 250 kDaand one of approximately 500 kDa) (lanes B and C,Fig. 2(1 and 2)). The protein pattern reveals the presence ofmultiple high molecular weight bands, some of themcatalytically active against MUA, so we focused our studyonly on complexes with acetyl esterase activity.

A combination with an SDS gel as second dimensionallowed the separation into subunits of the complexesobserved in the native first dimension. Lanes B, C and Dwere cut, denatured and coupled to a SDS-PAGE(Electronic supplementary material Fig. S3). In each gelseveral proteins were resolved between 20 and 120 kDa,indicating that the complex disintegrated after treatmentwith SDS in small protein subunits, thus ruling out thepresence of high molecular weight proteins that co-migratein native gels.

Protein complexes isolation and mass spectrometry analysis

Next, a separation of the complexes bearing acetyl esteraseactivity was attempted. Tandem gel filtration chromatogra-phy which enabled to distinguish different peaks of proteinsharbouring the activity (Electronic supplementary Fig. S1)was used. Seven fractions were partially isolated, threecorresponding to acetylated xylan culture samples (Cx1,Cx2 and Cx3), two from sugar beet pulp cultures (Cs1 andCs2) and two from corn cob supernatant (Cc1 and Cc2). A

Fig. 1 Blue Native on 4–12% polyacrilammide gradient gel. Lanes:M’, MW markers stained with Coomassie blue. M, same marker underUV light. A, Zymogram of sugar beet pulp supernatant using MUA assubstrate. B, Zymogram of the same supernatant concentrated 30-fold.C, Zymogram of precipitated fraction. B’, Concentrated supernatantstained with Coomassie blue

Fig. 2 Native electrophoresison gradient polyacrylamide gelof supernatants from differentcarbon sources. Carbon sources:A, glucose; B, sugar beet pulp;C, corn cob; D, acetylatedxylan. 1, Zymogram for acetylesterase activity. 2, Total proteinpatterns stained with SyproRuby. M, molecular weightstandards

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pre-calibration allowed an estimate their molecular weights.Fractions smaller than 200 kDa were discarded becausethey were unlikely to belong to any protein complex andprobably corresponded to free enzymes. Subsequently,the protein fractions with acetyl esterase activity werepooled for further characterization by immunotechniquesto determinate interactions between different and specificenzymes.

One complex from the sugar beet pulp culture superna-tant was purified using chromatography on Blue Sepharose,carboxymethyl cellulose and hydroxylapatite. Only oneband with acetyl esterase activity was observed in a BNelectrophoresis (Fig. 3). The same pool was separated bySDS-PAGE where at least six subunits of differentmolecular weight were observed. The bands were submittedto MS/MS identification, and the results are summarized inTable 2. Band A as well as band B correspond to a glucan1,4-α-glucosidase. β-1,6-glucanase, α-L-arabinofuranosi-dase and alcohol dehydrogenase, were also identified in

band B. Also glucan 1,4-α-glucosidase was identified inboth bands C and D. Band E is a cell wall protein and bandF is acetyl xylan esterase 2 from P. purpurogenum.

Subunit composition, antibody selection and coIPof complexes

Polyclonal antibodies against five important xylan degrad-ing enzymes from P. purpurogenum such as ABF 1 (anti-ABF1), ABF 3 (anti-ABF3), xylanase A (anti-XYLA),AXE I (anti-AXE1) and AXE II (anti-AXE2) wereemployed to detect the respective enzymes in the com-plexes. Fractions collected from the tandem gel filtrationchromatography were submitted to dot blot analysis in bothnative and denatured conditions.

Dot blots in native condition were used to find anappropriate antibody for coIP of intact complexes (positivesignal in native condition; Table 1). The dot blots indenatured condition allowed the identification of antibodiesto be used in Western blots after coIP (positive signal indenatured condition; Table 1) so as to recognize distinctenzyme partners in the same immunoprecipitated structure.The results are presented in Table 1. ABF 3 was identifiedin all fractions in native conditions, followed by AXE onepresent in the two complexes of sugar beet pulp, in onefrom corn cob and in the largest complex from acetylatedxylan. ABF one is present in both Cx2 and Cs2, AXE 2 isin Cx3 and xylanase A only in the complex of corn cob.

Based on the results of the dot blot experiments, coIPwas performed to further confirm the true existence of thecomplexes. As shown in Fig. 4, all of the complexes wereimmunoprecipitated with the same antibody (anti-ABF3)since this enzyme is present in all fractions analyzed by dotblot in native condition.

The precipitated complexes Cx2 and Cs2 reactedpositively with anti-ABF1. Similar results were obtainedfor complexes Cs1 and Cc2 with anti-AXE1 and Cs3 withanti-XYLA as shown in the Western blot analysis after coIPexperiments. On the other hand, the blots showed that thespecific bands match with the molecular weight previouslydescribed for the enzymes (Fig. 4).

Fig. 3 Protein composition of a purified complex. Purity of thecomplex was checked by Blue Native coupled to zymogram for acetylesterase activity. The subunits were separated by SDS-PAGE and theprotein bands (A–F) were cut and analyzed by MS/MS

Anti-ABF1 Anti-AXE1 Anti-AXE2 Anti-ABF3 Anti-XYLA

Dn Nt Dn Nt Dn Nt Dn Nt Dn Nt

Cx1 − − + − − − − + − −Cx2 + − − − − − + + − −Cx3 − − − − + + − + − −Cs1 − − + + − − − + − −Cs2 + − + + − − − + − −Cc2 − − + + − − − + − +

Table 1 Dot blot analysis of thesubunits in the complexesseparated by gel filtration

Dn denatured, Nt native

A plus sign indicates presenceand a minus sign indicatesabsence of the enzyme in thecomplexes

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Enzymatic hydrolysis of natural substratesby the multienzyme complexes

Wheat arabinoxylan was treated with the different com-plexes and the products of the hydrolysis were detected bythin layer chromatography (TLC; Electronic supplementarymaterial Fig. S2). Only one of the complexes coming fromthe acetylated xylan culture (Cx3) was active showingoligosaccharides but no xylose; the complex Cc2 comingfrom corn cob behaved the same way. No activity wasobserved with complexes Cs1 and Cs2 from the sugar beetpulp culture, in agreement with the chemical compositionof the carbon source (low xylose content). The observedpatterns indicate specificity in the hydrolysis of arabinoxylan:in the acetylated xylan culture only one of the three complexesis active (Cx3), showing that complex protein composition isheterogeneous and not random. It should also be noted thatthese samples showed high activity (as shown in TLC) afterseveral months of storage at 4°C.

Discussion

2D BN/SDS-PAGE separates protein complexes in twosteps. Firstly, in their native state, followed by a denaturingsecond dimension where they are decomposed in theirsubunits. This method has been used to analyze complexesin bacteria like Escherichia coli (Lasserre et al. 2006) andHelicobacter pylori (Pyndiah et al. 2007). We used it inculture supernatants of P. purpurogenum where we wereable to obtain catalytically active bands in the first native

dimension (Fig. 2(1)). By means of zymograms we assayedacetyl esterase activity and we detected fluorescence in thesupernatants coming from sugar beet pulp, corn cob andacetylated xylan culture supernatants. The negative controllane corresponds to a supernatant from a glucose-grownculture where no activity and very low protein weredetected. This sugar is a catabolite repressor of P.purpurogenum hemicellulases (Díaz et al. 2008). In super-natants from acetylated xylan cultures four active bands areobserved (Fig. 2(1), lane D). One of them is the active bandof highest size found in all conditions studied, with amolecular mass over 700 kDa that is stained very weakly(Fig. 2(2), lane D). Following in size, two activity bandscorrespond to the bands of the most intensive protein stain.There is a 20 kDa band that presents a strong esteraseactivity absent in the other culture conditions. It maybelong to acetyl xylan esterase two from P. purpurogenumbecause acetylated xylan is a carbon source that induces ahigh amount of acetyl esterase activity (Egaña et al. 1996).Sugar beet pulp and corn cob also induce two complexes ofsimilar size (between 250 and 500 kDa) and enzymeactivity. The protein pattern in corn cob cultures is themost diverse with more bands than the other conditions.However, most of them do not have acetyl esterase activityand probably are complexes of a different composition.

Prior to the second dimension run, the native gel was cutin strips that were denatured by boiling and treatment with2-mercaptoethanol and SDS. Proteins were separated bysize in a second gel. Thus, proteins that migrate together ata high molecular weight in the first dimension rangebetween 20 and 120 kDa in the second dimension. TheSDS-PAGE gel indicates that no high molecular weightproteins are present in the sample, and since in BN theproteins are separated by size, the results indicate thatproteins are truly associated with each other. In addition,since the pattern is different in the three culture conditionsstudied, this suggests that the carbon source inducesenzymes or protein complexes of different size andcomposition.

The purification of the complexes confirms theirexistence as well defined catalytically active structures.The analysis of one complex by nanoLC-ESI-MS/MSrevealed the presence of other enzymes not identified byantibodies or by enzymatic assays. It showed the presenceof glucanases (Table 2) such as glucan 1,4-α-glucosidasewith several peptides identified with a high Mowse score(Perkins et al. 1999). A 20 kDa band was identified as AXEII of P. purpurogenum which explains the esterase activityin the zymogram and the identification by antibodies in thecomplexes using dot blots. The purification of individualenzymes is at times technically cumbersome; this may beexplained by strong non-covalent interactions betweenthem. We used saline gradients to separate a complex and

Fig. 4 Western blot analysis of the coimmuneprecipitated complexesby anti ABF3. Cx2 and Cx3 fractions coming from acetylated xylan.Cs2 and Cs1 fractions coming from sugar beet pulp and Cc2, fractioncoming from corn cob

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Table 2 List of protein identified by mass spectrometry in the purified complex coming from sugar beet pulp supernatant

Band Namea Accession # % ofcoverage b

Molmassc

pI Scored Matchese Sequences

A gi|159128620glucan 1,4-α -glucosidase

EDP53734 44 67630 5.11 1337 18 VLVDLFR

SSGLSLSAR

ALVEGSTFAK

ALVEGSTFAKR

ALANHKVYTDSFR

IGSISITSTSLAFFK

ESDGSIVWESDPNR

KESDGSIVWESDPNR

DLTWSYAAFLTANMR

SVYAINSGIPQGAAVSAGR

DLTWSYAAFLTANMRR

YNVDMTAFTGAWGRPQRD

SYTVPAACGVSTATENDTWR.-

DIYSSAAVGTYASSTSTFTDIINAVK

SAKPGIIIASPSTSEPDYYYTWTR

TYADGYVSIVQAHAMNNGSLSEQFDK

VITEYVNSQAYLQTVSNPSGGLASGGLAEPK

SGKDANTVLASIHTFDPEAGCDDTTFQPCSPR

B gi|70988699 glucan1,4-α -glucosidase

XP_749206 28 67630 5.04 955 11 VLVDLFR

VYTDSFR

ALVEGSTFAK

ALVEGSTFAKR

IGSISITSTSLAFFK

SVYAINSGIPQGAAVSAGR

DLTWSYAAFLTANMRR

YNVDMTAFTGAWGRPQR

DIYSSAAVGTYASSTSTFTDIINAVK

VITEYVNSQAYLQTVSNPSGGLASGGLAEPK

SGKDANTVLASIHTFDPEAGCDDTTFQPCSPR

B gi|70983969 β-1,6-glucanase

XP_747510 22 51683 5.51 253 5 LMTPAGANFALMR

ILGSPWSAPGWMK

AASASAYCSNSAGNYK

QTIVGFGAAVTDATVTSFNTLSASVLQDLLNK

HTIGASDLSGDPAYTYDDNGGKADPSLSGFNLGDR

B gi|13991905α-L-arabinofuranosidase

AAK51551 9 52818 5.39 186 3 AYGVFISPGTGYR

HYNFGLLLNANDGTK

QFHEDATFCPQSGLSGQGNSIR

B gi|70998574 AlcoholDehydrogenase

XP_754009 6 37612 7.55 66 2 IFELMEQGK

QDGVEAIDFFAR

C gi|70988699 glucan1,4-α -glucosidase

XP_749206 5 67630 5.04 170 2 VYTDSFR

DIYSSAAVGTYASSTSTFTDIINAVK

D gi|70988699 glucan1,4-α -glucosidase

XP_749206 8 67630 5.04 198 3 ALVEGSTFAK

SVYAINSGIPQGAAVSAGR

SAKPGIIIASPSTSEPDYYYTWTR

D gi|70985723 endo-1,4-β -xylanase

XP_748367 3 33345 6.03 78 1 SSGTVTTANHFK

E gi|133920236 cellwall protein PhiA

CAM54066 27 19654 5.20 254 3 IGYTTGAQPAPR

QGWAIDSQNHLQFQGK

YFGIVAIHSGSAVQYQPFSAAK

152 Appl Microbiol Biotechnol (2011) 89:145–155

no effect was observed on its integrity since only one bandwas detected in a BN gel (Fig. 3).

The xylanosomes are discrete, multifunctional, multien-zyme complexes that degrade xylan (Deng et al. 2005);therefore the presence of xylan main chain degrading(endoxylanases and ß-xylosidases) and debranchingenzymes is needed. In the case of the purified complex,one xylanase and two debranching enzymes (AXE II andABF 1) were identified. This complex may thus beclassified as a xylanosome, but additional enzymatic assaysare required for further characterization.

The crude extracts were also subjected to tandem gelfiltration and the fractions were analyzed by dot blot usinga battery of antibodies. Native and denatured fractions gavedifferent information (Table 1). The antibody against ABF3reacted positively in native conditions in all of the fractionstested, but in denatured conditions the binding was poorexcept for Cx2. This pattern makes this antibody suitablefor recognition under native conditions as is the case ofcoIP. Anti-ABF1 binds to Cx2 and Cs2 in denaturedconditions but not under native conditions, probablybecause under the latter arabinofuranosidase one is part ofthese complexes but is located in a region where no epitopeis exposed. This interpretation is also valid for acetyl xylanesterase one in complex Cx1: a positive reaction is foundwhen the samples are boiled. Finally, the dot blots show theproteins that are present and absent in all complexesevaluated and the possible location of the components ofsome of them.

The products of coIP were analyzed by Western blotwhich implies that the complexes are denatured anddecomposed into their subunits that are then separated inSDS PAGE. For example, subunits such as AXE I interactwith ABF three in the fraction Cx1 (as shown by apositive reaction in Western blots (Fig. 4), but this is thelargest MW complex detected, indicating that probablythere are other protein members, since the low molecularweight of both enzymes alone do not fulfil ~700 kDa. These

results are strong additional evidence of the production ofmultienzyme complexes by P. purpurogenum.

These complexes are composed of enzymes specificallyinduced by the carbon source. Sugar beet pulp has a verylow concentration of xylose (Saulnier and Thibault 1999)so xylan is not expected to be present. This explains the factthat no hydrolysis of arabinoxylan is detected in TLC(Electronic supplementary material Fig. S2) when com-plexes produced in sugar beet pulp are evaluated. On theother hand, only one of the complexes from acetylatedxylan cultures hydrolysed arabinoxylan suggesting thateach complex may have a specialized function in thedegradation process.

P. purpurogenum is an active secretor of enzymes,including isoenzymes, and the pattern of expression ofthese proteins depends on the carbon source present in themedia (Chávez et al. 2006). This work shows that thecarbon source also affects the way in which proteinsinteract since the enzyme composition differs betweencomplexes in all of the conditions tested. Protein–proteininteractions may thus be a mechanism used by fungi toimprove catalysis and enzyme stability (Chang and Mahoney1995; Ellenrieder and Daz 1996; Mukhopadhyay et al.2003). Moreover, protein association protects enzymes frominhibition by heavy metals and other chemicals as shown inChaetomium sp., a termophilic fungus (Ohtsuki et al. 2005).These properties make these complexes valuable biotechno-logical tools.

Based on our results we propose that the secretedenzymes associate with each other in defined structuralunits. Their heterogeneous distribution is shown in thechromatographic and electrophoretic patterns. The interac-tions between subunits are specific and sufficiently strongsince even by precipitation with a high concentration ofammonium sulphate they appear unchanged after reconstitu-tion (dialyzed samples). Nevertheless, what remains unclear ishow the proteins associate; the interactions could be mediatedby direct protein–protein interactions as well as more

Table 2 (continued)

Band Namea Accession # % ofcoverage b

Molmassc

pI Scored Matchese Sequences

F gi|74626767Acetylxylanesterase 2

O59893 12 24033 6.26 88 1 AGLSYEVGTCAAGGFDQRPAGFSCPSAAK

M, N and Q represent oxidation in methionine (M) and deamidation in asparagine (N) or glutamine (Q), respectivelya Function annotations were retrieved from NCBInr (http://www.ncbi.nlm.nih.gov/)b The portion of the protein sequence that was observed by MS/MS analysis of the peptide mappingc The molecular mass or molecular weight of the protein identifiedd The threshold was set up by the server at the significance level P≥0.05 for random hit; scores greater than 52 were taken as a significant match based inMowse score (http://www.matrixscience.com)e The number of matching peptides to the target proteins

Appl Microbiol Biotechnol (2011) 89:145–155 153

indirectly by glycosylations. Besides, the possibility existsthat interactions may be mediated by extracellular polysac-charides (as it has been found in other microorganisms(Iwashita 2002)), by scaffolding proteins like dockerins incellulosomes (Levasseur et al. 2004) or by core proteins(Bayer et al. 1998). The protein pattern in the first dimensionof BN electrophoresis shows an absence of smearing, butinstead, the presence of several well defined bands. Thisindicates the existence of defined structures due to specificnon-random interactions. The protein complex architecture,whether its formation is intra- or extracellular, and thepotential existence of a genetic coordination remain unclear.

P. purpurogenum is able to adjust protein-proteininteractions to allow better adaptation to the environment.If we can understand in detail the structural conformationand the nature of the interactions in each complex, it will bepossible to prepare enzyme mixtures having increasedactivity and stability when used for defined biotechnolog-ical purposes.

Acknowledgements This work was supported in part by FONDE-CYT Grants Nº 1070368 and 1100084 and UNAB Grant DI-07-06/I.The authors wish to thank the Proteomics Core Facility at SD-BRIN(NIH Grant Number 2 P20 RR016479 from the INBRE Program ofthe National Center for Research Resources) for the mass spectrom-etry analysis. We also thank Dr. Barbara Goodman for a criticalreading of the manuscript.

References

Bayer EA, Chanz H, Lamed R, Shoham Y (1998) Cellulose, cellulaseand cellulosome. Curr Opin Struct Biol 8(5):548–557

Belancic A, Scarpa J, Peirano A, Diaz R, Steiner J, Eyzaguirre J(1995) Penicillium purpurogenum produces several xylanases:purification and properties of two of the enzymes. J Biotechnol41(1):71–79

Benoit I, Danchin EGJ, Bleichrodt R-J, De Vries RP (2007)Biotechnological applications and potential of fungal feruloylesterases based on prevalence, classification and biochemicaldiversity. Biotechnol Lett 30:387–396

Bollag DM, Edelstein SJ (1991) Protein methods. Wiley, New YorkBradford MM (1976) A rapid and sensitive method for the

quantification of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal Biochem 72:248–254

Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM,Carnemolla B, Orecchia P, Zardi L, Righetti PG (2004) Bluesilver: a very sensitive colloidal Coomassie G-250 staining forproteome analysis. Electrophoresis 25(9):1327–1333

Chang BS, Mahoney RR (1995) Enzyme thermostabilization bybovine serum albumin and other proteins: evidence for hydro-phobic interactions. Biotechnol Appl Biochem 22(2):203–214

Chávez R, Schachter K, Navarro C, Peirano A, Aguirre C, Bull P,Eyzaguirre J (2002) Differences in expression of two endoxyla-nase genes (xynA and xynB) from Penicillium purpurogenum.Gene 293:161–168

Chávez R, Bull P, Eyzaguirre J (2006) The xylanolytic enzyme systemfrom the genus Penicillium. J Biotechnol 123:413–433

Cosgrove DJ (2005) Growth of the plant cell wall. Nat Rev 6:850–861

de Ioannes P, Peirano A, Steiner J, Eyzaguirre J (2000) An a-L-arabinofuranosidase from Penicillium purpurogenum: produc-tion, purification and properties. J Biotechnol 76:253–258

de Vries RP, Visser J (2001) Aspergillus enzymes involved indegradation of plant cell wall polysaccharides. Microbiol MolBiol Rev 65:497

de Vries RP, Kester HCM, Poulsen CH, Benen JAE, Visser J (2000)Synergy between enzymes from Aspergillus involved in thedegradation of plant cell wall polysaccharides. Carbohydr Res327:401–410

Deng W, Jiang ZQ, Li LT, Wei Y, Shi B, Kusakabe I (2005) Variationof xylanosomal subunit composition of Streptomyces olivaceo-viridis by nitrogen sources. Biotechnol Lett 27:429–433

Díaz J, Chávez R, Larrondo LF, Eyzaguirre J, Bull P (2008)Functional analysis of the endoxylanase B (xynB) promoter fromPenicillium purpurogenum. Curr Genet 54(3):133–141

Egaña L, Gutierrez R, Caputo V, Peirano A, Steiner J, Eyzaguirre J(1996) Purification and characterization of two acetyl xylanesterases from Penicillium purpurogenum. Biotechnol ApplBiochem 24:33–39

Ellenrieder G, Daz M (1996) Thermostabilization of naringinase fromPenicillium decumbens by proteins in solution and immobiliza-tion on insoluble proteins. Biocatal Biotransfor 14(2):113–123

Faulds CB, Mandalari G, Lo Curto RB, Bisignano G, ChristakopoulosP, Waldron KW (2006) Synergy between xylanases fromglycoside hydrolase family 10 and family 11 and a feruloylesterase in the release of phenolic acids from cereal arabinoxylan.Appl Microbiol Biotechnol 71:622–629

Fritz M, Ravanal MC, Braet C, Eyzaguirre J (2008) A family 51 a-L-arabinofuranosidase from Penicillium purpurogenum: purification,properties and amino acid sequence. Mycol Res 112:933–942

Gal L, Pages S, Gaudin C, Belaich A, Reverbel-Leroy C, Tardif C,Belaich JP (1997) Characterization of the cellulolytic complex(cellulosome) produced by Clostridium cellulolyticum. ApplEnviron Microbiol 63:903

Gold ND, Martin VJJ (2007) Global view of the Clostridiumthermocellum cellulosome revealed by quantitative proteomicanalysis. J Bacteriol 189:6787–6795

Gordillo F, Caputo V, Peirano A, Chavez R, Van Beeumen J,Vandenberghe I, Claeyssens M, Bull P, Ravanal MC, Eyzaguirre J(2006)Penicillium purpurogenum produces a family 1 acetyl xylanesterase containing a carbohydrate-binding module: characteriza-tion of the protein and its gene. Mycol Res 110:1129–1139

Hidalgo M, Steiner J, Eyzaguirre J (1992) Beta-glucosidase fromPenicillium purpurogenum: purification and properties. BiotechnolAppl Biochem 15(2):185–191

Iwashita K (2002) Recent studies of protein secretion by filamentousfungi. J Biosci Bioeng 94:530–535

Jiang H, Chen D, Zhao P, Li Y, Zhu K (2005) Characterization of anovel, ultra-large xylanolytic complex (xylanosome) from E-86.Enzyme Microb Technol 36:923–929

Jiang Z, Dang W, Yan Q, Zhai Q, Li L, Kusakabe I (2006) Subunitcomposition of a large xylanolytic complex (xylanosome) fromStreptomyces olivaceoviridis E-86. J Biotechnol 126:304–312

Karboune S, Geraert P-A, Kermasha S (2008) Characterization ofselected cellulolytic activities of multi-enzymatic complex systemfrom Penicillium funiculosum. J Agric Food Chem 56:903–909

Laemmli UK (1970) Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 227:680–685

Lasserre JP, Beyne E, Pyndiah S, Lapaillerie D, Claverol S, Bonneu M(2006) A complexomic study of Escherichia coli using two-dimensional blue native/SDS polyacrylamide gel electrophoresis.Electrophoresis 27(16):3306–3321

Lee J (1997) Biological conversion of lignocellulosic biomass toethanol. J Biotechnol 56:1–24

154 Appl Microbiol Biotechnol (2011) 89:145–155

Lee YE, Lowe SE, Zeikus JG (1993) Regulation and characterizationof xylanolytic enzymes of Thermoanaerobacterium saccharolyticumB6A-RI. Appl Environ Microbiol 59:763–771

Leschot A, Tapia RA, Eyzaguirre J (2002) Efficient Synthesis of 4-methyl-umbelliferyl dihydroferulate. Synth Commun 32(20):3219–3223

Levasseur A, Pages S, Fierobe HP, Navarro D, Punt P, Belaich JP,Asther M, Record E (2004) Design and production in aspergillusniger of a chimeric protein associating a fungal feruloyl esteraseand a clostridial dockerin domain. Appl Environ Microbiol70:6984–6991

Mandels M, Weber J (1969) Production of cellulases. Adv Chem Ser95:391–414

Mo B, Callegari E, Telefont M, Renner KJ (2006) Proteomic analysisof the ventromedial nucleus of the hypothalamus (pars lateralis)in the female rat. (Translated from eng) Proteomics 6(22):6066–6074, in eng

Mukhopadhyay A, Hazra PP, Sengupta T, Saha R, Nandi R, SenguptaS (2003) Protein-protein interaction conferring stability to anextracellular acetyl (xylan) esterase produced by Termitomycesclypeatus. Biotechnol Prog 19:720–726

Musalem S, Steiner J, Contreras O (1984) Producción de celulasas porhongos aislados de maderas y suelos del sur de Chile. Bol Micol2:17–25

Ohtsuki T, Suyanto YS, Ui S, Mimura A (2005) Production of largemultienzyme complex by aerobic thermophilic fungus Chaeto-mium sp. nov. MS-017 grown on palm oil mill fibre. Lett ApplMicrobiol 40:111–116

Pason P, Kyu KL, Ratanakhanokchai K (2006) Paenibacilluscurdlanolyticus Strain B-6 Xylanolytic-cellulolytic enzyme sys-tem that degrades insoluble polysaccharides. Appl EnvironMicrobiol 72:2483–2490

Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-basedprotein identification by searching sequence databases using massspectrometry data. (Translated from English) Electrophoresis 20(18):3551–3567 (in English)

Polizeli MLTM, Rizzatti ACS, Monti R, Terenzi HF, Jorge JA,Amorim DS (2005) Xylanases from fungi: properties andindustrial applications. Appl Microbiol Biotechnol 67:577–591

Pyndiah S, Lasserre JP, Menard A, Claverol S, Prouzet-Mauleon V,Megraud F, Zerbib F, Bonneu M (2007) Two-dimensional bluenative/SDS gel electrophoresis of multiprotein complexes fromHelicobacter pylori. Mol Cell Proteomics: MCP 6:193–206

Ravanal MC, Callegari E, Eyzaguirre J (2010) A novel bifunctional{alpha}-L-arabinofuranosidase/xylobiohydrolase (ABF3) fromPenicillium purpurogenum. Appl Environ Microbiol 76(15):5247–53

Saha BC (2003) Hemicellulose bioconversion. J Ind MicrobiolBiotechnol 30:279–291

Saulnier L, Thibault JF (1999) Ferulic acid and diferulic acids ascomponents of sugar-beet pectins and maize bran heteroxylans. JSci Food Agric 79:396–402

Sigoillot C, Camarero S, Vidal T, Record E, Asther M, Perez-BoadaM, Martinez MJ, Sigoillot JC, Colom JF, Martinez AT (2005)Comparison of different fungal enzymes for bleaching high-qualitypaper pulps. J Biotechnol 115:333–343

Steiner J, Socha C, Eyzaguirre J (1994) Culture conditions forenhanced cellulase production by a native strain of Pennicilliumpurpurogenum. World J Microbiol Biotechnol 10:280–284

Tabka M, Herpoelgimbert I, Monod F, Asther M, Sigoillot J (2006)Enzymatic saccharification of wheat straw for bioethanolproduction by a combined cellulase xylanase and feruloylesterase treatment. Enzyme Microb Technol 39:897–902

Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer ofproteins from polyacrylamide gels to nitrocellulose sheets:procedure and some applications. PNAS 76:4350–4354

Waeonukul R, Kyu KL, Sakka K, Ratanakhanokchai K (2008) Effectof carbon sources on the induction of xylanolytic-cellulolyticmultienzyme complexes in Paenibacillus curdlanolyticus strainB-6. Biosci Biotechnol Biochem 72:321–328

Williamson G, Kroon P, Faulds C (1998) Hairy plant polysaccharides:a close shave with microbial esterases. Microbiology 144:2011–2023

Wittig I, Braun H-P, Schägger H (2006) Blue native PAGE. Nat Protoc1:418–428

Yamin TT, Ayala JM, Miller DK (1996) Activation of the native 45-kDa precursor form of interleukin-1-converting enzyme. J BiolChem 271(22):13273–13282

Appl Microbiol Biotechnol (2011) 89:145–155 155