An Acidic Thermostable Recombinant Aspergillus nidulans Endoglucanase Is Active towards Distinct Agriculture Residues

Hindawi Publishing CorporationEnzyme ResearchVolume 2013 Article ID 287343 10 pageshttpdxdoiorg1011552013287343

Research ArticleAn Acidic Thermostable Recombinant Aspergillus nidulansEndoglucanase Is Active towards Distinct Agriculture Residues

Eveline Queiroz de Pinho Tavares1 Marciano Regis Rubini1

Thiago Machado Mello-de-Sousa1 Gilvan Caetano Duarte1

Fabriacutecia Paula de Faria2 Edivaldo Ximenes Ferreira Filho1

Cynthia Maria Kyaw1 Ildinete Silva-Pereira1 and Marcio Jose Poccedilas-Fonseca3

1 Department of Cellular Biology Institute of Biological Sciences University of Brasilia 70910-900 Brasilia DF Brazil2 Department of Biochemistry and Molecular Biology Institute of Biological Sciences Federal University of Goias 74001-970 GoianiaGO Brazil

3 Department of Genetics and Morphology Institute of Biological Sciences University of Brasilia ET 1825Darcy Ribeiro University Campus 70910-900 Brasilia DF Brazil

Correspondence should be addressed to Marcio Jose Pocas-Fonseca mpossasunbbr

Received 19 March 2013 Revised 11 June 2013 Accepted 13 June 2013

Academic Editor Joaquim Cabral

Copyright copy 2013 Eveline Queiroz de Pinho Tavares et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Aspergillus nidulans is poorly exploited as a source of enzymes for lignocellulosic residues degradation for biotechnologicalpurposes This work describes the A nidulans Endoglucanase A heterologous expression in Pichia pastoris the purification andbiochemical characterization of the recombinant enzyme Active recombinant endoglucanase A (rEG A) was efficiently secreted asa 35 kDa protein which was purified through a two-step chromatography procedure The highest enzyme activity was detected at50∘CpH 4 rEG A retained 100 of activity when incubated at 45 and 55∘C for 72 h Purified rEG A kinetic parameters towards

CMC were determined as 119870119898

= 275 plusmn 433mgmL 119881max = 1185 plusmn 011mmolmin and 558 IU (international units)mg specificactivity Recombinant P pastoris supernatant presented hydrolytic activity towards lignocellulosic residues such as banana stalksugarcane bagasse soybean residues and corn straw These data indicate that rEG A is suitable for plant biomass conversion intoproducts of commercial importance such as second-generation fuel ethanol

1 Introduction

One of the major challenges of modern society is to promoteeconomic growth in a sustainable model Global demandsof energy consumption stimulate the research on alternativefuels aiming the reduction of the dependence on non-renewable energy sources For some decades now Braziland the USA have successfully produced bioethanol fromsugarcane and corn respectively Nonetheless plant biomassgenerated by extensive cultures and which is not totallyconverted into useful by-products such as fertilizers andanimal feed tends to accumulate and cause environmentalproblems Numerous efforts have been made in order to

develop biotechnological routes to produce the so-calledsecond-generation bioethanol from agriculture residues suchas corn stover rice straw sorghum bagasse corncobs wheatbran wheat straw and sugarcane bagasse The limiting stepof this process is the availability of low-cost efficient enzymesto convert lignocellulose into fermentable glucose units

Filamentous fungi can produce and secrete enzymeswhich efficiently degrade cellulose a linear polymer of glu-copyranose units connected by 120573-14 bonds to oligosaccha-rides and glucose Based onmodel organisms from the generaTrichoderma and Phanerochaete fungi cellulolytic enzymesacting in synergism have been classified as (1) endoglu-canases or endo-120573-14-glucanases (EC 3214) responsible

2 Enzyme Research

for the random attack of internal glycosidic bonds of thecellulose amorphous region generating oligosaccharides ofvarious sizes and new chain ends for the action of a secondclass of enzymes (2) cellobiohydrolases (EC 32191) whichprocessively degrade the reducing and nonreducing ends ofamorphous or crystalline cellulose regions releasing glucoseor cellobiose and (3) 120573-glucosidases which hydrolyze cel-lobiose and other small oligosaccharides into glucose Morerecently swollenins which are proteins homologous to plantexpansins were reported to cooperate in cellulose hydrolysisby fungi such as T reesei Trichoderma pseudokoningiiTrichoderma asperellum and Aspergillus fumigatus

Members of the genus Aspergillus have been described asefficient cellulases producers Aspergillus nidulans producesthree endoglucanases four cellobiohydrolases and one 120573-glucosidase Lockington et al [1] demonstrated that like inmany other cellulolytic fungi A nidulans cellulase genesexpression is regulated by the carbon and the nitrogensources In a cocultivation study involving the bacterium Pec-tobacterium carotovorum a proteomic approach revealed thatA nidulans was the main responsible for leave litter decom-position [2] Saykhedkar et al [3] have recently demonstratedthat A nidulans produces and secretes a complete set ofenzymes capable of degrading cellulose hemicelluloses andpectin present in sorghum stover without chemical pretreat-ment of this substrateThese data point out toA nidulans as acandidate for plant biomass conversion at the industrial level

A nidulansEndoglucanaseA (EGA) genewas cloned andcharacterized it comprises a 1228 bp sequence interruptedby four introns [4] The corresponding enzyme presented35 kDa displayed the highest activity at 50∘CpH 65 andretained 50 of activity when incubated at 30ndash70∘C for 1 h

Aiming the production of high levels ofA nidulans EGAwhich could allow a refined biochemical characterization ofthis potential industrial biocatalyst in this workwe expressedA nidulans eglA cDNA in the Pichia pastoris heterologoussystem P pastoris has been widely described as a robust andefficient producer of recombinant proteins which are secretedto the culture supernatant The purified recombinant EG A(rEG A) showed the highest CMCase activity at 50∘C and pH4 It also displayed a remarkable thermostability retainingalmost 100 of activity after 48 h of incubation at the opti-mum temperature range Purified rEG A kinetic parameterstowards CMC were also determined Furthermore we coulddetect the release of reducing sugars from the incubationof the P pastoris recombinant strain crude extract withagricultural wastes such as banana stalk sugarcane bagassesoybean residues and corn strawThese features indicate thatA nidulans rEG A is suitable to biotechnological processessuch as second-generation biofuel production

2 Materials and Methods

21 Microorganisms and Growth Conditions Conidia (106mL) of theA nidulans pabaA1 biA1methG1 and argB strainwere inoculated in Pontecorvorsquos minimal medium (MM)supplemented with 15 gL hydrolyzed casein 10 gL glucose2 gL peptone and 05 gL yeast extract and incubated at

30∘C with agitation for 24 h Mycelia were then washed withdistilled water and inoculated in MM enriched with 1 gLball-milled steam-exploded sugarcane bagasse (SCB) for 24 h(30∘C200 rpm) for cellulase genes induction Sugar canebagasse was obtained from the Jardinopolis Alcohol andSugarMill (JARDEST Sao Paulo Brazil) and was prepared bytreatment with superheated steam followed by instantaneousdecompression in a reactor system as described by Kling etal (1987) [5] Processed SCB samples were kept at 4∘C

The P pastoris GS115 (his4) strain was used as heterolo-gous host according to the conditions described in the PichiaExpression kit (Invitrogen Carlsbad CA USA)

For cloning experiments and plasmid manipulationsEscherichia coli XL10Gold TetrD (119898119888119903119860)183 D(119898119888119903119862119861-ℎ119904119889119878119872119877-119898119903119903)173 1198901198991198891198601 11990411990611990111986444 119905ℎ119894-1 1199031198901198881198601 11989211991011990311986096 1199031198901198971198601119897119886119888 Hte [F1015840 119901119903119900119860119861 119897119886119888119868q11988511986311987215 Tn10 (Tetr) Amy Camr](Stratagene La Jolla CA USA) was used Bacteria weregrown at 37∘C in LB medium [5 gL yeast extract 10 gLpeptone and 10 gL NaCl] supplemented with the appropriateantibiotics when necessary

22 Synthesis Cloning and Expression of the A nidulans eglAcDNA After induction for cellulase genes A nidulans totalRNA was extracted using the Trizol reagent (Invitrogen)Synthesis and amplification of the eglA cDNA wereperformed by RT-PCR using the specific primers EGA-SnaBI (51015840-TACGTAGCTTTCACATGGTTTGG-31015840) andEGA-AvrII (51015840-CCTAGGTTATTGACTTCCCACG-31015840)whose design was based on the eglA gene sequence describedby Chikamatsu et al [4] (accession no AB009402)The underlined bases indicate the restriction enzymesrecognition sites A 12 kb cDNA molecule was amplifiedand cloned into the pGEM-T vector (Promega Madison -WI) After transformation of E coli XL10 Gold competentcells the eglApGEM-T plasmid DNA was extracted anddigested with SnaBI and AvrII in order to be transferred tothe P pastoris pPIC9 expression vector In this plasmidialconstruct named eglApPIC9 the A nidulans eglA cDNAwas placed under control of the methanol-inducible AOX1promoter in frame with the Saccharomyces cerevisiae 120572-factorsignal peptide encoding sequence (Pichia Expression KitCarlsbad CA USA) After DNA sequencing confirmationthe eglApPIC9 plasmid was used to transform the P pastorisGS115 strain (Invitrogen Carlsbad CA USA) according tothe supplierrsquos recommendations One hundred transformantclones were grown and screened for efficient EG A-enzymesecretion as previously described [6]

The recombinant clone presenting the highest CMCaseactivity and one negative control (aP pastoris clone harboringthe empty pPIC9 vector) were selected for further analyses

23 Purification of the Recombinant EG A from the P pastorisCulture Supernatant An isolated colony of the P pastorisrecombinant strain harboring the eglApPIC9 construct wasgrown in 100mL of BMGY medium [100mM potassiumphosphate pH 50 134 gL YNB (Invitrogen) 00004 gLbiotin 10mLL glycerol] in 1-L flasks incubated at 30∘C at250 rpm until the culture reached an OD

600value of 10 Cells

Enzyme Research 3

were then harvested and washed two times with distilledwater resuspended in 100mL of BMMYmedium in 1-L flasksand incubated under the same conditions for additional 48 hwith the addition of methanol to a final concentration of05 (vv) at every 24 h in order to maintain the inductioncondition Finally cells were centrifuged at 12000 g4∘C for15min the supernatant was collected and 02 gL sodiumazide was added

The rEG A purification procedure was based on twosystems of ultrafiltration membranes followed by a two-step chromatographic protocol Initially the supernatant wasapplied into an ultrafiltration system employing a membranewith molecular weight cut-off of 50000Da (Biomax-50NMWL Millipore) under pressure of 25 kgfcm2 at 10∘CThe MW50 eluted fraction was concentrated on a MW10ultrafiltration membrane with the molecular weight cut-off of 10000Da (Biomax-10 NMWL Millipore) and thensubmitted to gel filtration chromatography in a SephadexG50 column (600 times 27 cm) equilibrated with 05M sodiumphosphate buffer pH 70 25mM NaCl at a 20mLh flux at28∘C The eluted fractions were tested for CMCase activityand protein concentration (A280 nm) Fractions presentingCMCase activity were then pooled dialyzed (Dialysis tubingD9402 Sigma Aldrich) and applied onto an ionic exchangecolumn (Q-Sepharose 150 times 25 cm) previously equilibratedwith 05M sodium phosphate buffer pH 70 at a 20mLhflux at 28∘CThe eluted fractions displaying CMCase activitywere pooled and employed for the recombinant enzymebiochemical characterization

24 rEG A SDS-PAGE and Zymogram Analyses The SDS-PAGE protocol was performed according to Sambrook andRussel [7] employing 12 (wv) polyacrylamide gel followedby coomassie blue R250 or silver nitrate staining [8] In orderto detect enzyme activity a zymogram assay was performedon a 12 (wv) polyacrylamide gel containing 015 (wv)CMC (carboxymethylcellulose sodium salt low viscositySigma) as previously described [9] Prior to the zymogramanalysis the samples were precipitated with 10 TCA andwashed twice with cold 100 acetone

25 rEG A Biochemical Characterization TheCMCase activ-ity employing CMC as substrate was determined by themethod described by Mandels et al [10] and modified byFilho et al [11] Analyzed samples consisted of the culturemedium supernatant (crude extract CE) and the purifiedrEG A obtained as described previously The activity valuescorrespond to the means of three independent experimentsin three technical replicates The statistical analysis wasperformed using ANOVA with 5 level of significance andthe SPSS for Windows version 170 program

The amount (mgmL) of reducing sugars produced ineach reaction was determined by the DNS method [12]measured by spectrophotometry at A540 nm (SpectramaxM2

e (Mol Dev Corp Sunnyvale CA USA)) using glucoseas standard One unit of enzyme activity was established asthe amount of enzyme that released 1120583mol of reducing sugarper minute per mL expressed as IUmL

Enzyme activity was evaluated at temperatures rangingfrom 30 to 80∘C Optimal pH was established with thefollowing buffers 50mM sodium acetate (pH 40ndashpH 65)50mM sodium phosphate (pH 60ndashpH 70) and 50mMTris-Cl (pH 65ndashpH 80)The determined optima temperature andpH were employed in the subsequent experiments

The evaluation of the rEG A thermostability was per-formed by enzyme preincubation at 45∘C 55∘C 70∘C and80∘C for 3 12 24 48 and 72 h

The effect of metal ions and other chemicals on theendoglucanase activity was assayed by the addition to thereaction system of 18mM (the HgCl

2concentration which

causes 50 inhibition of the rEG A CMCase activity) ofthe following reagents AlCl

3 CaCl

2 ZnSO

4 NaCl CoCl

2

CuCl2 KCl FeCl

3 EDTA SDS beta-mercaptoethanol and

14-dithio-DL-threitol (DTT)rEG A substrate specificity was performed using

CMC filter paper (Whatman no 1 6 cm times 1 cm straps)xylan microcrystalline cellulose (Avicel Sigma) and p-ni-trophenyl-120573-D-glucoside (pNPG) as substrates The finalconcentration of reducing sugars was determined asdescribed previously In order to evaluate the rEG A activitytowards the substrate 4-methylumbelliferyl-120573-D-cellobioside(MUC) a qualitative analysis was performed employing UVlight to detect the fluorescent digestion product

In all experiments the values for CMCase activity repre-sent the averages of experimental triplicate

26 Determination of rEG A Kinetic Parameters To deter-mine the rEG A Michaelis-Menten kinetic parameters (119870

119898

and119881max) CMC (concentration ranging from0 to 35mgmL)was employed as substrate in a reaction mixture containing775 120583g purified protein in 50mM sodium acetate pH 40 at50∘C for 30min The obtained data were analyzed using theprogram EnzFitter Windows (Biosoft Cambridge UK)

27 Enzyme Activity towards Agricultural Residues rEG Acapacity to hydrolyze lignocellulosic substrates derived fromagriculture was assayed in 50mL flasks containing 23 ofsubstrate solution [03mL 10M sodium acetate buffer pH40 40mg of the substrate (banana stem ball-milled steamexploded sugarcane bagasse soybean cultivation waste orcorn stover) and 37mL distilled water] and 13 of P pastorisCE (125UmL FPAse activity) at the proportions of 25 5075 and 100 in the final volume of 6mL completed withdistilled water Sodium azide (02 gL) was added to avoidcontamination by microorganisms Reaction mixtures wereincubated for 24 48 72 and 96 h at 50∘C150 rpm Aliquotsof 05mL were periodically collected

3 Results

31 Cloning of the A nidulans eglA cDNA and Productionof the Recombinant Enzyme The RT-PCR assay using totalRNA from A nidulans grown with 10 gL SCB as the solecarbon source produced a 12 kb cDNA fragment compatibleto the size predicted from the splicing of the four putative

4 Enzyme Research

CE

70

55

40

35

25

17

rEGA(kDa)

(a)

CErEGA

(b)

Figure 1 Electrophoretic profile of the recombinant endoglucanase by SDS-PAGE 12 (wv) The gels were stained with brilliant bluecoomassie (a) and activity gel with Congo red (b) MMmolecular massmarker (Fermentas) in kDa rEGA sample eluted fromQ-Sepharosecolumn CE crude extract

Table 1 Purification of the recombinant endoglucanase from the supernatant of P pastoris

Fractions Total protein (120583g) Recombinant endoglucanase A activityTotal activity (IU) Specific activity (IUmg) Yield () Purification fold (x)

Crude extract 3669 11694 32 100 1Concentrated fraction (MW50) 1997 945 05 ND NDUltrafiltered fraction (MW50) 2135 7704 36 659 11Concentrated fraction (MW10) 1251 395 03 34 01Ultrafiltered fraction (MW10) 2466 8957 36 ND NDSephadex G50 031 433 141 37 44Dialyzed 036 412 115 35 36Q-Sepharose 004 240 558 21 175ND not determined

introns [4]This cDNA fragment was cloned into the pGEM-T vector and then transferred to the P pastoris pPIC9expression vector under control of the inducible promoterAOX1

Based on the highest CMCase a P pastoris recombinantclone was selected for the next experiments One cloneharboring the empty pPIC9 vector was used as negativecontrol

P pastoris clones were grown under induction conditionsand culture supernatants were evaluated for enzyme activityduring a 120 h period The P pastoris clone containing theeglApPIC9 construct presented the highest CMCase activityfrom 24 h of growth the same activity was maintainedthroughout the cultivation period No enzyme activity wasdetected for the negative control

32 Purification of rEG A The recombinant P pastoris strainwas grown upon induction for 48 h and the supernatant wasapplied into a ultrafiltration system employing a membrane

with a molecular weight cut-off of 50000Da (Biomax-50 NMWL Millipore) followed by a cut-off membraneof 10000Da (Biomax-10 NMWL Millipore) The obtainedsample was named CONCMW10 and was subsequentlypurified by a two-step separation protocol After passagethrough a gel filtration column an isolated peak of CMCaseactivity distinct from the one presenting the highest proteinconcentration was obtained (data not shown) Samples cor-responding to this activity peak were pooled and submittedto ionic exchange chromatography which resulted in a sharppeak (data not shown) corresponding to a single protein bandof 35 kDa coincident with the CMC degradation spot in theactivity gel (Figure 1)

The four fractions produced by the purification protocolwere assayed for CMCase activity and protein concentrationEach fraction specific activities and recovery yield of therecombinant enzyme after the purification steps are summa-rized in Table 1 After purification rEGA specific activity wasdetermined as 558 IUmL

Enzyme Research 5

00

01

02

03

04

05

Crude extract

a

a

a b

d

e

a c

a b c a b c

b cbb c d

a dActiv

ity (U

Im

L)

30 40 50 60 70 80

rEGA

Temperature (∘C)

Figure 2 Effect of temperature on the crude extract and rEG Aenzyme activity on CMC The points on the graphs represent theaverage of experimental triplicates and the vertical bars their stan-dard deviation The different letters indicate statistical differencesbetween the different assays in the same fraction (119875 lt 005)

Table 2 Effect of treatment with different agents (chelators metalions detergents and reducing agents) on rEG A activity

Treatment Relative activity ()Control 10000 plusmn 395SDS 3124 plusmn 473lowast

EDTA 10369 plusmn 407DTT 13243 plusmn 311lowast

FeCl3sdot6H2O 9200 plusmn 609AlCl3 6710 plusmn 454lowast

CaCl2 11255 plusmn 691lowast

ZnSO4 8303 plusmn 719lowast

NaCl 9714 plusmn 430CoCl2sdot7H2O 13226 plusmn 319lowast

CuCl2 12037 plusmn 233lowast

KCl 9934 plusmn 420120573-mercaptoethanol 18181 plusmn 1109lowast

Asterisks (lowast) indicate statistical difference within the same fraction (119875 lt005) when compared to control The results are presented in terms ofactivity plusmn standard deviation The endoglucanase activity was assayed afterthe addition of 18mM of the agents to the reaction system

33 Temperature and pH Effect on rEG A Activity CE andrEG A enzyme activities were analyzed in temperaturesranging from 30 to 80∘C at pH 65 (Figure 2) For bothsamples the highest CMCase activity was observed whenreaction proceeded at 40ndash60∘C At the extreme temperatures(30 and 80∘C) enzyme activity was 50 lower

Enzyme activity towards CMC was assayed from pH 3 topH 9 Optimum pH was around 40 for both enzyme prepa-rations (Figure 3) Alkalinization of the reaction mixture ledto a marked decrease in CMCase activity

Preincubation of the reaction mixture at 45 and 55∘C forup to 72 h did not significantly affect enzyme activity On

the other hand temperatures of 70∘C and 80∘C provoked asevere decrease in CMC hydrolysis from the beginning of thepreincubation period (Figure 4)

34 Effect of Metal Ions and Other Chemicals on the rEG AActivity The effect of cations chelants and reducing agentson the purified rEG A activity was assayed (Table 2) All thereagents were tested at 18mM since this concentration ofHgCl2led to a 50 inhibition of the rEG A activity rEG A

CMCase activity was inhibited in 70 by SDS The reducingagents DTT and beta-mercaptoethanol increased enzymeactivity by 32 and 81 respectively EDTA did not affect rEGA activity

35 Substrate Specificity of rEG A Whatman no 1 filterpaper microcrystalline cellulose (Avicel Sigma) xylan fromoat spelts (Sigma) p-nitrophenyl-beta-D-glucopyranoside(pNPG Sigma) and 4-methyl-beta-umbelliferyl D-cello-bioside (MUC Sigma)were employed for the rEGA substratespecificity assay (Figure 5) Filter paper activity represented50 of the activity towards the common endoglucanasesubstrate CMC while microcrystalline cellulose and xylanhydrolysis efficiency corresponded to 20 of the verified forthis substrate The recombinant enzyme showed no activitytowards pNPG and MUC (data not shown) Recombinant Ppastoris strain CE presented a significant FPase activity

36 rEG A Kinetic Parameters Increasing CMC concentra-tions were employed for the determination of the A nidulanspurified recombinant endoglucanase 119870

119898and 119881max values

With aid of the EnzFitter program rEG A 119870119898

and 119881maxvalues were determined as 275 plusmn 433mgmL and 1185 plusmn

011mmolmin respectively

37 Enzyme Activity towards Agricultural Residues P pas-toris recombinant strain CE was assayed for the capacityof hydrolyzing the natural substrates banana stem ball-milled steam-exploded sugarcane bagasse soybean residuesand corn stover Aliquots from the reaction mixtures werecollected after 24 48 72 and 96 h of incubation andreleased total reducing sugars (TRSs) were quantified The72 h incubation period was identified as the most efficientfor the release of TRS when the rEG A CE was addedat the proportion of 100 (Figure 6) Corn stover was thelignocellulosic substrate more susceptible to enzyme hydrol-ysis 250120583gmL of TRS a value significantly higher whencompared to other agriculture residues TRSs released frombanana stem sugarcane bagasse and soybean residues werein the range of 200120583gmL In terms of hydrolysis percentageTRS value corresponded to 387 of the corn stover masspresent in the assay

4 Discussion

The major impediment for an economically feasible second-generation bioethanol production is the development ofstrategies to break down the chemical bonds of the polysac-charides that tightly form the cell wall thus producing free

6 Enzyme Research

3 4 5 6 6 65 7 7 8 900

01

02

03

04

05

06

Sodium acetateSodium phosphateTris-Cl

Buffer

aa

bc

c cc e

c d

c e c e

pH

Enzy

mat

ic ac

tivity

(IU

mL)

(a)

Sodium acetateSodium phosphateTris-Cl

Buffer

b

3 4 5 6 6 65 7 7 8 900

01

02

03

04

05

06

a

a

pH

aa

b cb c b c c

b c

Enzy

mat

ic ac

tivity

(IU

mL)

(b)

Figure 3 Enzymatic activity of recombinant endoglucanase rEGA (a) and crude extract (b) onCMCThebuffers usedwere Tris-HCl sodiumacetate or sodium phosphate at the final concentration of 50mM The columns represent the averages of experimental triplicate with theircorresponding standard deviation The different letters indicate statistical differences between the different assays (119875 lt 005)

0 3 12 24 48 720

20

40

60

80

100

120

140

45∘C

55∘C 80

∘C70

∘C

aa

a a a a

c

d b

TemperatureTime (h)

Resid

ual a

ctiv

ity (

)

(a)

45∘C

55∘C 80

∘C70

∘C

Temperature

0 3 12 24 48 720

20

40

60

80

100

120

140

a

a a a

a

a

c

b

Time (h)

Resid

ual a

ctiv

ity (

)

(b)

Figure 4 Thermostability of the recombinant endoglucanase crude extract (a) and rEG A The results are expressed in terms of residualenzymatic activity ()The points represent the averages of experimental triplicate with their corresponding standard deviationThe differentletters indicate statistical differences between the different assays (119875 lt 005)

sugars that could be fermented by the already standardizedprotocols employing S cerevisiae In this view the formu-lation of enzyme cocktails which could efficiently degradecellulose and hemicellulose themajor components of naturalresidues such as sugarcane bagasse and corn straw is strategi-cally important In order to achieve an optimized hydrolysisrate glycosyl hydrolases prospection and characterizationpipelines should be guided by the feedstock composition

Although refined data on sugarcane bagasse composition arenot available sugarcane leaves and culms present about 30cellulose 10 pectins and 50 hemicelluloses [13] Such aheterogeneous composition which can vary according to thesoil characteristics and cultivation conditions justifies theprospection for new enzymes presenting distinct biochem-ical properties such as peculiar substrate specificities anddifferent optima temperature and pH In this view enzyme

Enzyme Research 7

0

50

100

150

200

250

Crude extract

a a

b

c

d

bb

c

Rela

tive a

ctiv

ity (

)

CMC

Avic

el FP

Xyla

n

Substrate

rEGA

Figure 5 Substrate specific activity of the recombinant endoglu-canase Crude extract and rEG A on carboxymethylcellulose(CMC) microcrystalline cellulose (Avicel) filter paper (FP) andxylanThedifferent letters indicate statistical differences between thedifferent assays (119875 lt 005) 100 activity towards CMC as substratecorresponds to 028 IUmL

0

50

100

150

200

250

300

Banana stalkSugar cane bagasse

Soybean residuesCorn straw

a a b a

b

Natural substrates

Tota

l red

ucin

g su

gars

(120583g

mL)

Figure 6 Distinct natural lignocelluloses residues degradation in 72hoursThe experiments were performed in triplicate using the crudeextract of P pastoris recombinant The results are expressed as totalsugar formed using the DNS method The columns represent theaverages of experimental triplicate with their corresponding stan-dard deviation The different letters indicate statistical differencesbetween the different assays (119875 lt 005)

diversity is of pivotal importance to the design of enzymecocktails capable of converting sugar cane residues in usefulby-products at the industrial level

In this work we have cloned the A nidulans endoglu-canase A cDNA in P pastoris The heterologous host wasable to produce and to secrete the enzyme in its activeform upon induction with 05 methanol rEG A maximum

activity was achieved within 24 h of induction and it wasmaintained up to 120 h which is advantageous for severalindustrial applications This result also validates the heterol-ogous expression approach since we achieved a large scaleof enzyme production in a short period of time In nativesecretion systems or even in heterologous expressionmodelsother studies reported the maximum cellulase activity in thesupernatant after longer periods of induction [14ndash16]

Subsequently we have performed a two-step ultrafiltra-tion followed by a two-step chromatography purificationprocedure The first ultrafiltration step resulted in a sample(UFMW50) presenting a CMCase specific activity morethan seven times higher than the concentrated one (CON-CMW50) The same effect was not observed for the secondultrafiltration probably because the enzyme passed throughthe membrane pore The ability of CMCase to penetrate anultrafiltrationmembranemay be due to its compact structureandor nonuniformity of membrane pore size

The chromatographic step consisted in gel filtrationwhich separated two distinct enzymatic peaks (data notshown) In the fractions present in the first peak specificactivity value increased almost 45 times (Table 1) The overallrecovery level and fold purification of rEG A were 21 and175 respectively The low yield value was mainly due to theloss of enzyme activity in the ultrafiltration step

rEG A optimal temperature range (40ndash60∘C Figure 2)indicates that it corresponds to a mesophilic enzyme [14]Mesophilic endoglucanases are useful in several biotechno-logical processes such as in the formulation of biostoningand biopolishing agents for the textile industry Further-more rEG A maintained 100 of the enzyme activity aftera 48 h preincubation period at 45 and 50∘C (Figure 4)Thermostability was also described for other recombinantfungal endoglucanases produced in P pastoris [16ndash18] andthis characteristic is important for industrial purposes sincethe enzyme can work efficiently for long periods withoutrequiring addition of more enzymes to the process

The optimum pH (40) we described for rEG A does notmatch the value (pH 65) observed for the partially purifiednative EGA [4] Some endoglucanases fromAspergilli displayhigher activity in the acidic pH range [19 20] Accordingto Hahn-Hagerdal et al [21] and Dashtban et al [22]acidophilic enzymes are more suitable to industrial lignocel-lulose degradation since most of the substrate is pretreatedwith inorganic acids

The reducing agents 120573-mercaptoethanol and DTT pro-voked 80 and 32 increase in enzyme activity respectively(Table 2) this suggests that disulfide bonds are not of pivotalimportance in rEG A three dimensional structure stabi-lization EDTA did not significantly affect rEG A functionpossibly indicating that it is not a metalloprotein as it isthe case of Aspergillus terreus strains M11 and DSM 826endoglucanases which are inhibited by this ions chelator[20 23] As it was reported for other fungal endoglucanases[24ndash27] A nidulans rEG A interaction with Ca2+ Co2+ andCu2+ resulted in increased enzyme activity possibly becausethese ions exert a stabilizing effect on the enzyme structurewithout interfering in the catalytic site

8 Enzyme Research

rEG A was able to degrade CMC and to a lesser extentfilter paper (Figure 5) Some other studies have also reportedthe activity of fungal endoglucanases towards filter paper[23 24 27] which would represent a better substrate forcellobiohydrolases The ability to hydrolyze different sub-strates can be explained by nonspecific bindings in the activesite andor by the presence of distinct catalytic domainseach one presenting a particular activity [28] Our researchgroup has recently demonstrated that theHumicola grisea varthermoidea recombinant cellobiohydrolase 12 produced inP pastoris acts as a bifunctional enzyme presenting activitytowards crystalline and amorphous cellulose [18] Such dualenzymes can be particularly useful to several bioconversionprocessesA nidulans rEGA presented no significant activitytowards pNPG xylan andAvicel Interestingly in the recom-binant P pastoris CE we detected both FPase and xylanaseactivities this is possibly due to unspecific enzyme activitiespresent in the host secretome by itself andor interaction withthe recombinant endoglucanase [29 30] Salinas et al [31]detected a background CMCase activity in the supernatantof a P pastoris recombinant strain harboring an emptyexpression vector the authors attributed such an activityto P pastoris genomic sequences encoding for glycosidehydrolases

The specific activity we have determined for A nidulansrEG A (558 IUmg) is higher than the value observed foranother fungal endoglucanase expressed in P pastoris forbiotechnological purposes the Trametes versicolor recombi-nant enzyme (35ndash40 IUmg) [31] When compared to indus-trial enzymes whose data are normally not available due topatents confidentiality rEG A specific activity towards CMCwas 2 times higher than Spezyme 3 (Genencor Intl) andmore than 15 times higher than Biocellulase A (Quest Intl)[32]These data corroborates rEGA as a potential biocatalyst

The recombinant P pastoris CE was assayed for thedegradation of natural lignocellulosic biomass banana stemsteam-exploded sugar cane bagasse soybean residue andcorn stover The degradation of residues by holocellulasesis an efficient and inexpensive process to obtain productswith high added value such as the ones derived from pulpand paper industry second-generation biofuels compostingfood and feed among others [33] However these residuesrecalcitrance hampers the access of hydrolytic enzymes inorder to release monomeric sugars especially glucose tobe fermented in subsequent processes [34 35] rEG A bestdegradation efficiency occurred for corn stover The differenthydrolysis efficacy presented for the distinct plant biomassresidues possibly reflects the complexity of the lignocellulosecomposition and its structural arrangement According toMansfield et al [36] the efficacy of enzyme complexes tohydrolyze natural substrates is linked to the innate structuralcharacteristics of the substrate andor to the modificationsthat occur during the pretreatment or the saccharificationsteps

rEG A activity towards different agriculture residues mayalso be related to the lignin content of these substrates cellwall According to Howard et al [37] lignin represents 15

of rice straw and corn cobs 30ndash40 of nut shells and 25ndash35 of softwood stems biomass Lignin is possibly the mainresponsible for the apparently low percentage of corn stovermass conversion (387 which corresponds to 025 gL) intoreducing sugars by rEG A

In addition degradation efficiency by rEG A enzymecould be optimized by different pretreatment schemes orandthe association of other enzymes such as cellobiohydrolasesand xylanases in the context of enzyme cocktails

Although native A nidulans endoglucanase A had beenpartially characterized in a previous study [4] most of thefeatures described here for the recombinant enzyme wereunknownThus this work provided novel andmore completeinformation about an endoglucanase with biotechnologicalpotential due to the optimum temperature range the acidicoptimumpH the thermostability and the capacity to degradeeven nonpretreated natural residues such as corn stover

Conflict of Interests

There is no conflict of interests for any of the authors on thispaper

Authorsrsquo Contribution

EvelineQueiroz de Pinho Tavares andMarciano Regis Rubinicontributed equally to this work

Acknowledgments

This work was funded by FINEPMCT (Bioethanol Net-work) CNPq (National Council for Scientific and Techno-logical Development-Brazil) and FAP-DF (Research SupportFoundation of the Federal District Brazil)

References

[1] R A Lockington L Rodbourn S Barnett C J Carter and JMKelly ldquoRegulation by carbon and nitrogen sources of a family ofcellulases in Aspergillus nidulansrdquo Fungal Genetics and Biologyvol 37 no 2 pp 190ndash196 2002

[2] T Schneider B Gerrits R Gassmann et al ldquoProteome analysisof fungal and bacterial involvement in leaf litter decomposi-tionrdquo Proteomics vol 10 no 9 pp 1819ndash1830 2010

[3] S Saykhedkar A Ray P Ayoubi-Canaan S D Hartson RPrade andA JMort ldquoA time course analysis of the extracellularproteome of Aspergillus nidulans growing on sorghum stoverrdquoBiotechnology for Biofuels vol 5 no 52 pp 1ndash17 2012

[4] G Chikamatsu K Shirai M Kato T Kobayashi and NTsukagoshi ldquoStructure and expression properties of the endo-120573-14-glucanase A gene from the filamentous fungusAspergillusnidulansrdquo FEMS Microbiology Letters vol 175 no 2 pp 239ndash245 1999

[5] S H Kling C Carvalho Neto M A Ferrara J C R TorresD B Magalhaes and D D Y Ryu ldquoEnhancement of enzymatichydrolysis of sugar cane bagasse by steam explosion pretreat-mentrdquoBiotechnology and Bioengineering vol 29 no 8 pp 1035ndash1039 1987

[6] M R Rubini A J P Dillon C M Kyaw F P Faria M JPocas-Fonseca and I Silva-Pereira ldquoCloning characterization

Enzyme Research 9

and heterologous expression of the first Penicillium echinulatumcellulase generdquo Journal of Applied Microbiology vol 108 no 4pp 1187ndash1198 2010

[7] J Sambrook andDWRusselMolecular CloningmdashALaboratoryManual Cold Spring Harbor Laboratory Press New York NYUSA 3rd edition 2001

[8] H Blum H Beier and H J Gross ldquoImproved silver stainingof plant proteins RNA and DNA in polyacrilamide gelsrdquoElectrophoresis vol 8 no 2 pp 93ndash99 1987

[9] X Sun Z Liu Y Qu and X Li ldquoThe effects of wheatbran composition on the production of biomass-hydrolyzingenzymes by Penicillium decumbensrdquo Applied Biochemistry andBiotechnology vol 146 no 1ndash3 pp 119ndash128 2008

[10] M Mandels R Andreotti and C Roche ldquoMeasurement ofsaccharifying cellulaserdquo Biotechnology and Bioengineering Sym-posium no 6 pp 21ndash33 1976

[11] E X F Filho J Puls and M P Coughlan ldquoBiochemical char-acteristics of two endo-120573-14-xylanases produced by Penicilliumcapsulatumrdquo Journal of Industrial Microbiology vol 11 no 3 pp171ndash180 1993

[12] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959

[13] A P de Souza D C C Leite S Pattathil M G Hahn andM SBuckeridge ldquoComposition and structure of sugarcane cell wallpolysaccharides implications for second-generation bioethanolproductionrdquoBioEnergy Research vol 6 no 2 pp 564ndash579 2013

[14] S E Chaabouni T Mechichi F Limam and N MarzoukildquoPurification and characterization of two low molecular weightendoglucanases produced by Penicillium occitanis mutant Pol6rdquo Applied Biochemistry and Biotechnology vol 125 no 2 pp99ndash112 2005

[15] O RibeiroMWiebeM Ilmen L Domingues andM PenttilaldquoExpression of Trichoderma reesei cellulases CBHI and EGI inAshbya gossypiirdquo Applied Microbiology and Biotechnology vol87 no 4 pp 1437ndash1446 2010

[16] J Thongekkaew H Ikeda K Masaki and H Iefuji ldquoAnacidic and thermostable carboxymethyl cellulase from theyeast Cryptococcus sp S-2 purification characterization andimprovement of its recombinant enzyme production by highcell-density fermentation of Pichia pastorisrdquo Protein Expressionand Purification vol 60 no 2 pp 140ndash146 2008

[17] Y Bai R Guo H Yu L Jiao S Ding and Y Jia ldquoCloningof endo-120573-glucanase I gene and expression in Pichia pastorisrdquoFrontiers of Agriculture in China vol 5 no 2 pp 196ndash200 2011

[18] G S Oliveira C J Ulhoa M H L Silveira et al ldquoAn alkalinethermostable recombinant Humicola grisea var thermoideacellobiohydrolase presents bifunctional (endoexoglucanase)activity on cellulosic substratesrdquoWorld Journal of Microbiologyand Biotechnology vol 29 no 1 pp 19ndash26 2013

[19] S K Garg and S Neelakantan ldquoEffect of cultural factors oncellulase activity and protein production by Aspergillus terreusrdquoBiotechnology and Bioengineering vol 24 no 1 pp 109ndash1251982

[20] J Gao H Weng Y Xi D Zhu and S Han ldquoPurificationand characterization of a novel endo-120573-14-glucanase from thethermoacidophilic Aspergillus terreusrdquo Biotechnology Lettersvol 30 no 2 pp 323ndash327 2008

[21] B Hahn-Hagerdal M Galbe M F Gorwa-Grauslund GLiden and G Zacchi ldquoBio-ethanolmdashthe fuel of tomorrow fromthe residues of todayrdquoTrends in Biotechnology vol 24 no 12 pp549ndash556 2006

[22] M Dashtban H Schraft and W Qin ldquoFungal bioconversionof lignocellulosic residues Opportunities amp perspectivesrdquo Inter-national Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009

[23] AM ElshafeiMMHassan BMHarounOMAbdel-FatahH M Atta and A M Othman ldquoPurification and properties ofan endoglucanase of Aspergillus terreus DSM 826rdquo Journal ofBasic Microbiology vol 49 no 5 pp 426ndash432 2009

[24] A Karnchanatat A Petsom P Sangvanich et al ldquoA novelthermostable endoglucanase from the wood-decaying fungusDaldinia eschscholzii (EhrenbFr) Rehmrdquo Enzyme and Micro-bial Technology vol 42 no 5 pp 404ndash413 2008

[25] S-Y Liu M A Shibu H-J Jhan C-T Lo and K-C PengldquoPurification and characterization of novel glucanases fromTrichoderma harzianum ETS 323rdquo Journal of Agricultural andFood Chemistry vol 58 no 19 pp 10309ndash10314 2010

[26] D Liu R Zhang X Yang et al ldquoExpression purification andcharacterization of two thermostable endoglucanases clonedfrom a lignocellulosic decomposing fungiAspergillus fumigatusZ5 isolated from compostrdquo Protein Expression and Purificationvol 79 no 2 pp 176ndash186 2011

[27] A Nazir R Soni H S Saini R K Manhas and B S ChadhaldquoPurification and characterization of an endoglucanase fromAspergillus terreus highly active against barley 120573-glucan andxyloglucanrdquo World Journal of Microbiology and Biotechnologyvol 25 no 7 pp 1189ndash1197 2009

[28] J S van Dyk and B I Pletschke ldquoA review of lignocellulose bio-conversion using enzymatic hydrolysis and synergistic cooper-ation between enzymesmdashfactors affecting enzymes conversionand synergyrdquo Biotechnology Advances vol 30 pp 1458ndash14802012

[29] L R Lynd P J Weimer W H van Zyl and I S PretoriusldquoMicrobial cellulose utilization fundamentals and biotechnol-ogyrdquoMicrobiology andMolecular Biology Reviews vol 66 no 3pp 506ndash577 2002

[30] D Mattanovich A Graf J Stadlmann et al ldquoGenome secre-tome and glucose transport highlight unique features of the pro-tein production host Pichia pastorisrdquo Microbial Cell Factoriesvol 8 article 29 2009

[31] A SalinasMVegaM E Lienqueo AGarcia R Carmona andO Salazar ldquoCloning of novel cellulases from cellulolytic fungiheterologous expression of a family 5 glycoside hydrolase fromTrametes versicolor in Pichia pastorisrdquo Enzyme and MicrobialTechnology vol 49 no 6-7 pp 485ndash491 2011

[32] R A Nieves C I Ehrman W S Adney R T Elander andM E Himmel ldquoSurvey and analysis of commercial cellulasepreparations suitable for biomass conversion to ethanolrdquoWorldJournal ofMicrobiology and Biotechnology vol 14 no 2 pp 301ndash304 1998

[33] C Sanchez ldquoLignocellulosic residues biodegradation and bio-conversion by fungirdquo Biotechnology Advances vol 27 no 2 pp185ndash194 2009

[34] M G Adsul J E Ghule R Singh et al ldquoPolysaccharides frombagasse applications in cellulase and xylanase productionrdquoCarbohydrate Polymers vol 57 no 1 pp 67ndash72 2004

[35] M G Adsul J E Ghule H Shaikh et al ldquoEnzymatic hydrolysisof delignified bagasse polysaccharidesrdquo Carbohydrate Polymersvol 62 no 1 pp 6ndash10 2005

[36] S D Mansfield C Mooney and J N Saddler ldquoSubstrateand enzyme characteristics that limit cellulose hydrolysisrdquoBiotechnology Progress vol 15 no 5 pp 804ndash816 1999

10 Enzyme Research

[37] R L Howard E Abotsi E L J van Rensburg and S HowardldquoLignocellulose biotechnology issues of bioconversion andenzyme productionrdquoAfrican Journal of Biotechnology vol 2 no12 pp 602ndash619 2003

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Enzyme Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

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Volume 2013

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Advances in

Virolog y

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

BioMed Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2013

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Marine Biology

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

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