production of recombinant trichoderma reesei

Research ArticleProduction of Recombinant Trichoderma reeseiCellobiohydrolase II in a New Expression System Based onWickerhamomyces anomalus

Dennis J Diacuteaz-Rincoacuten1 Ivonne Duque1 Erika Osorio1

Alexander Rodriacuteguez-Loacutepez12 Angela Espejo-Mojica1 Claudia M Parra-Giraldo3

Rauacutel A Poutou-Pintildeales4 Carlos J Almeacuteciga-Diacuteaz1 and Balkys Quevedo-Hidalgo4

1 Institute for the Study of Inborn Errors of Metabolism Facultad de Ciencias Pontificia Universidad Javeriana Bogota Colombia2Departamento de Quımica Facultad de Ciencias Pontificia Universidad Javeriana Bogota Colombia3Unidad de Proteomica y Micosis Humanas Grupo de Enfermedades Infecciosas Departamento de MicrobiologıaFacultad de Ciencias Pontificia Universidad Javeriana Bogota Colombia4Grupo de Biotecnologıa Ambiental e Industrial (GBAI) Departamento de Microbiologıa Facultad de CienciasPontificia Universidad Javeriana Bogota Colombia

Correspondence should be addressed to Carlos J Almeciga-Dıaz cjalmecigajaverianaeducoand Balkys Quevedo-Hidalgo bquevedojaverianaeduco

Received 12 May 2017 Revised 19 July 2017 Accepted 2 August 2017 Published 30 August 2017

Academic Editor Sunney I Chan

Copyright copy 2017 Dennis J Dıaz-Rincon et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Cellulase is a family of at least three groups of enzymes that participate in the sequential hydrolysis of cellulose Recombinantexpression of cellulases might allow reducing their production times and increasing the low proteins concentrations obtained withfilamentous fungi In this study we describe the production of Trichoderma reesei cellobiohydrolase II (CBHII) in a native strainofWickerhamomyces anomalus Recombinant CBHII was expressed inW anomalus 54-A reaching enzyme activity values of up to145U Lminus1The enzyme extract showed optimum pH and temperature of 50ndash60 and 40∘C respectively Enzyme kinetic parameters(119870119872of 273mM and Vmax of 231 120583Mminminus1) were between the ranges of values reported for other CBHII enzymes Finally the

results showed that an enzymatic extract of W anomalus 54-A carrying the recombinant T reesei CBHII allows production ofreducing sugars similar to that of a crude extract from cellulolytic fungiThese results show the first report on the use ofW anomalusas a host to produce recombinant proteins In addition recombinant T reesei CBHII enzyme could potentially be used in thedegradation of lignocellulosic residues to produce bioethanol based on its pH and temperature activity profile

1 Introduction

Cellulases are enzymes from the glycoside hydrolase family(EC 321-) that are expressed by a broad spectrumof bacteriaand fungi strains [1 2] Among these microorganisms white-and brown-rot fungi are considered the major producers ofcellulases including Trichoderma sp Aspergillus sp Schizo-phyllum commune and Volvariella volvacea [1 2] Cellulaseis a family of at least three groups of enzymes endo-(14)-120573-D-glucanase (EC 3214 EG) exo-(14)-120573-D-glucanase (EC32191 CBH) and120573-glucosidases (EC 32121 BG) [3]These

enzymes act sequentially on the cellulose hydrolysis (1) EGrandomly attacks on O-glycosidic bonds resulting in glucanchains of different lengths (2) CBH acts on glucan chainsends to release 120573-cellobiose and (3) BG hydrolyzes the 120573-cellobiose to produce glucose [3]

Trichoderma reesei is one of themost important cellulasesproducing filamentous fungi since it contains the coreenzymes necessary for the complete hydrolysis of lignocellu-lose material [4] Among these enzymes cellobiohydrolaseshave been shown to be one of the most important compo-nents within this process [4] Enzymes involved in hydrolysis

HindawiEnzyme ResearchVolume 2017 Article ID 6980565 8 pageshttpsdoiorg10115520176980565

2 Enzyme Research

of cellulose polymer molecules have been recombinantlyexpressed [5] which might allow reducing the produc-tion times and increasing the low proteins concentrationsobtained with filamentous fungi Recombinant expressionmight also facilitate the scaling up of the enzyme productionas well as the implementation of Simultaneous Saccharifica-tion and Fermentation (SSF) and Separate Hydrolysis andFermentation (SHF) processes Saccharomyces cerevisiae hasbeen used for the production of recombinant CBH fromPhanerochaete chrysosporium [6] and Talaromyces emersonii[7] while T reesei CBHII has been expressed in S cerevisiae[8 9] P pastoris [10 11] and Y lipolytica [11]

Conventional microbial systems (eg E coli S cerevisiaeand P pastoris) have allowed the production of a long listof recombinant proteins [12] However there is a growinginterest in the identification of new hosts that allow the pro-duction of high-quality or cost-efficient recombinant proteins[13] Recently a native strain of Wickerhamomyces anomaluswas isolated from sugar cane bagasse at Puerto Lopez MetaColombia (unpublished results) W anomalus is a yeast thathas been isolated from different sources [14] and its reportedapplications include food biopreservation bioremediationand production of phytases and biofuels [14 15] However tothe best of our knowledge there are no reports showing itsuse as host to produce recombinant proteins

In this paper we describe the production of recombinantcellobiohydrolase II (CBHII EC 32191) from T reesei usingthe native strainW anomalus 54-A Furthermore the effectsof pH and temperature on enzyme activity were character-ized as well as the kinetic properties of the enzyme andits application on the hydrolysis of a lignocellulose materialOverall the results showed the potential ofW anomalus 54-A as host to produce recombinant proteins and showed thatrecombinant T reesei CBHII has similar characteristics tothose reported for the wild-type enzyme

2 Materials and Methods

21Microorganism CultureMedia andVectors W anomalus54-A was previously isolated from sugar cane bagasse atPuerto Lopez Meta Colombia Initially the microorgan-ism was identified by carbon assimilation profile using theAPI 20C AUX kit (bioMerieux Marcy lrsquoEtoile France)Further microorganism identification was done by ampli-fication of internal transcribed spacer (ITS) using theprimers ITS1 51015840-TCCGTAGGTGAACCTGCGG-31015840 and ITS451015840-TCCTCCGCTTATTGATATGC-31015840 and Pfu DNA poly-merase (Thermo Fisher Scientific Inc San Jose CA US)undermanufacturer instructions Identity ofWanomalus 54-A was finally confirmed byMALDI-TOF analysis (Unidad deInvestigacion en Proteomica y Micosis Humana PontificiaUniversidad Javeriana)

Plasmid pKS2-ST (Dualsystems Biotech SchlierenSwitzerland) was used as expression vector In this vectorprotein expression is regulated by the alcohol dehydrogenaseII promoter (EC 1111 ADH2) while the secretion signalof the Saccharomyces cerevisiae invertase SUC2 mediatesthe secretion of the recombinant protein Escherichiacoli DH5 was used for cloning purposes which was

cultured in Luria-Bertani (LB) medium supplemented with100mgmLminus1 ampicillin Yeast cultures were performed inYPD supplemented with 500mgmLminus1 geneticin for theselection of clones and 60mgmLminus1 for protein expressioncultures

22 Expression Vector and Recombinant Strains Cellobio-hydrolase II gene (cbhII) from T reesei (GenBank NumberGU7247631) was previously codon-optimized for its expres-sion in S cerevisiae by GeneArt (Thermo Fisher ScientificInc) (unpublished data) The sequence encoding for theCBHII signal peptide was identified by using SignalP [16]and removed from the gene sequence Comparison of thecodon usage tables of S cerevisiae and W anomalus showeda similar profile between both microorganisms (Supplemen-tary Figure 1 in Supplementary Material available online athttpsdoiorg10115520176980565) Two codons (CGC andCGG) which encode Arg (14 residues 3 in CBHII) showedsignificant differences between both microorganisms Never-theless the other two Arg codons have a comparable usagebetween S cerevisiae and W anomalus In this sense thiscodon optimized sequence was used for the CBHII expres-sion in W anomalus 54-A Codon optimized cbhII gene wasinserted downstream of the SUC2 secretion signal in the vec-tor pKS2-ST to produce pKS2-STCBHII (SupplementaryFigure 2)The pKS2-STCBHII vector was used to transformcompetent cells of W anomalus 54-A by electroporationusing the Gene Pulser electroporation system Xcell (Bio-Rad Laboratories) at 1400V and 200Ω The W anomalus54-A clones were selected in YPD medium supplementedwith 500mgmLminus1 geneticin Clones were confirmed by PCRusing the primers 51015840-GAGGAGAGCATAGAAATGGGG-31015840and 51015840-CAGCAGTAGCCATAGCACCA-31015840 which amplify afragment from cbhII gene The PCR-positive clones wereevaluated at 55mL scale according to vector pKS2-ST man-ufacturerrsquos instructions (Dualsystems Biotech) Briefly eachclone was incubated for 10 h in 10mL YPD after which 15mLof fresh YPD medium was added After 14 h incubation30mL of fresh YPD medium was added to reach a finalculture volume of 55mL After 6 h of incubation it wasexpected that the glucose was exhausted and this timewas considered as the beginning of the induction phase1mL aliquots were taken every 24 h for 96 h to measureextracellular enzyme activity and cell density All the assayswere performed in triplicate at 28∘C and 180 rpm Residualglucose quantitation was carried out by DNS method [17]Crude extracellular fractions of the clone with the highestenzyme activity were loaded and processed by SDS-PAGE

23 Evaluation of Carbon Sources The clone that showedthe highest enzyme activity was used to evaluate the effectof carbon source (ie glucose and glycerol) on the enzymeproduction Cultures were carried out in 2 (wv) tryptoneand 1 (wv) yeast extract supplemented with glyceroland glucose in different concentrations 2 (wv) glucose1 (wv) glucose 1 (wv) glycerol 2 (wv) glucose 1(wv) glycerol and 2 (wv) glycerol All cultures weresupplemented with 60mgmLminus1 geneticin

Enzyme Research 3

24 Culture at 24 L Scale The W anomalus 54-A clone thatshowed the highest CBHII activity at 55mL scale was scaledup to 24 L in a 37 L Bioengineering KFL2000 bioreactorCultures were done in modified YPD medium [2 (wv)tryptone 1 (wv) yeast extract 1 (wv) glucose and 1(wv) glycerol] supplemented with 60mgmLminus1 geneticinBriefly 2mL from the cell bank was inoculated into 18mL ofculture medium and incubated for 24 h at 28∘C and 180 rpmThe preinoculum was used to inoculate 180mL of freshculture medium and incubated for 24 h at 28∘C and 180 rpmFinally the inoculum was used to inoculate 1000mL of freshculture medium at the bioreactor After 15 h culture 1200mLof fresh medium was added to reach a final volume of 24 Land cultured during additional 96 h at 28∘C 400 to 800 rpmand pH 60

25 Cellobiohydrolase Activity Assay TheCBHII activity wascarried out as previously described [18 19] using 5mMp-nitrophenyl 120573-D-cellobioside (pNPC Sigma-Aldrich) insodium acetate buffer 50mM pH 50 One enzyme unit wasdefined as the amount of enzyme releasing 1 120583mol of p-nitrophenol per min and the activity was expressed as U Lminus1

26 Characterization of Recombinant CBHII The CBHIIenzyme extract was evaluated at different pH and temper-ature conditions using pNPC To evaluate the effect of pHand temperature on enzyme activity the enzyme activityassay was performed at 30 40 50 60 70 and 80 plusmn02 and at 30 40 50 60 and 70∘C for 1 h The reactionswere stopped and read as described above Apparent kineticparameters (119870

119872and Vmax) were estimated for the crude

extract by using pNPC between 0 and 65mM and fitting theexperimental data to a Michaelis-Menten model using thesoftware GraphPad PRISM 60

27 Chrysanthemum Wastes Degradation Assay The effectof recombinant CBHII on cellulose hydrolysis was evaluatedby using Chrysanthemum wastes as previously described[18 19] with modifications Chrysanthemum wastes wereautoclaved at 121∘C for 15min Experiments were performedin substrate submerged cultures carried out in 100mL flasksin a rotatory shaker at 150 rpm and 30∘C for 20 daysFlasks contained 1 Chrysanthemum wastes in 40mL of thefollowing enzyme crude extracts (1) cellulolytic and hemicel-lulolytic extract from a Penicillium sp culture (hereafter Ce-Hem extract) (2) concentrated recombinant CBHII extractfromW anomalus 54-A (hereafter rCBHII) and (3) a 1 1 Ce-Hem rCBHII extracts mixture Control culture was carriedout by using 40mLof distilledwater All cultures were carriedout with 10mMsodium azide 01 tween 80 and pH 50Theresponse variable was the concentration of reducing sugars asmeasured by DNS method [17] Results are reported as g Lminus1of reducing sugars after subtraction of the results obtainedwith the control cultures and normalized by the initial unis ofCBHII present in each extract Production of Ce-Hem extractwas carried out by using a Penicillium sp strain (Collectionof Microorganisms of Pontificia Universidad Javeriana) Forthis purpose 20mL of a 106 conidia mLminus1 suspension was

inoculated in 200mL of a rice straw medium without nitro-gen source and incubated for 8 days at 28∘C and 120 rpmThecrude extract was obtained by centrifugation and sequentialfiltrations through Whatman paper Number 42 and 045and 022 120583m polyether sulphone membranes (Pall Corp PortWashington NY USA) Concentrated recombinant CBHIIextract from W anomalus 54-A was produced by using a24 L culture of the W anomalus 54-A Celo-32 clone asdescribed above Culture medium was centrifuged at 8000119892and filtered sequentially throughWhatman paperNumber 42and 045 and 022 120583m polyether sulphone membranes (PallCorp) Permeate was ultrafiltered through a 30 kDa cut-offmembrane (Millipore Billerica MA USA) until reaching afinal volume of 20mL CBHII activity in both extracts wasassayed as described above The 120573-glucosidase (BG) activityin both extracts was assayed using 5mM 4-nitrophenyl 120573-D-glucuronide (Sigma-Aldrich) as previously described [1819] One BG unit was defined as the amount of enzymereleasing 1 120583mol of p-nitrophenol per min and the activitywas expressed as U Lminus1 The endoglucanase (EG) activity inboth extracts was assayed using 2 carboxymethylcellulose(Sigma-Aldrich) [18 19] and the DNS method for quanti-tation of reducing sugars [17] One EG unit was defined asthe amount of enzyme releasing 1 120583mol of reducing sugarsequivalent to glucose per min [20] and the activity wasexpressed as U Lminus1

28 Statistical Analysis Differences between groups weretested for statistical significance by using one-way ANOVAAn error level of 5 (119901 lt 005) was considered significantAll analyses were performed using the software GraphPadPRISM 60

3 Results and Discussion

31 Production of Recombinant CBHII in W anomalus 54-AIn this study we evaluated the production of the recombinantCBHII in the native yeast strain 54-A previously isolatedfrom sugar cane bagasse from Puerto Lopez Meta Colom-bia This strain showed a carbon assimilation profile (API20C AUX bioMerieux) different from that observed for Scerevisiae strains and it was identified as Pichia anomala(recently renamed as Wickerhamomyces anomalus [21]) witha 945 ID and 025 T value Identity of this strain wasfurther confirmed as W anomalus through ITS amplifica-tion (GenBank accession number KX676490 Supplemen-tary Figure 3) and MALDI-TOF analysis (SupplementaryFigure 4)W anomalus is a yeast that have been isolated fromdifferent sources such as food- and feed-related systemsinsects and human clinical samples This yeast has a widerobustness to environmental stresses conditions like extremepH or low water activity [14] Biotechnological applicationsof W anomalus strains include biopreservation agent due toantimicrobial activity against variety of microorganisms infruits and cereals and production of enzymes (eg phytases)biofuels and bioremediation [14 15] However to the best ofour knowledge there are no reports of the use of this yeast asa host to produce recombinant proteins

4 Enzyme Research

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0 20 40 60 80

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tivity

(UL

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Celo-32Empty vector

(a)

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a)

21

66

3145

97116

200

92 Ｂ0 Ｂ

(b)

Figure 1 Recombinant T reeseiCBHII produced inW anomalus 54-A (a) PCR-positive clones ofW anomalus 54-A were evaluated at 55mLscale and the activity of recombinant T reesei CBHII was assayed in the extracellular fraction Among the four PCR-positive clones onlyWanomalus 54-A Celo 32 clone showed CBHII activity No activity was observed inW anomalus 54-A transfected with the empty vector Eachclone was evaluated in triplicate (b) Crude extracellular extracts fromW anomalus 54-A Celo 32 at 0 and 92 h were evaluated by SDS-PAGEfollowed by silver staining The arrow indicates the recombinant CBHII

Four clones were obtained after transformation of Wanomalus 54-A with the vector pKS2-STCBHII (Celo-22Celo-32 Celo-42 and Celo-51) which were confirmed byPCR The ADH2 gene promoter is repressed in the presenceof glucose since this metabolite inhibits the expression ofthe alcohol dehydrogenase regulator protein (Adr1) that is anactivator of theADH2 geneOn the other hand under glucosedepletion conditions the expression of Adr1 is increasedleading to the induction of theADH2 gene [22] In this sensewe first determined the residual glucose during the culture ofW anomalus 54-A pKS2-STCBHII clones to establish thebeginning of the induction phase We observed for all theevaluated clones that at the final culture step the glucosewasconsumed within the first 6 h of incubation (SupplementaryFigure 5) In this sense this point was considered as thebeginning of the induction phase which agrees with previousreports of the production of recombinant proteins in yeastunder the control of theADH2 gene promoter [22 23] Underthese culture conditions extracellular CBHII activity wasonly detected in W anomalus 54-A Celo-32 clone reachinga final enzyme activity of 57U Lminus1 after 96 h of induction(Figure 1(a)) As expected CBHII activity was not detectedinW anomalus 54-A transfected with the empty vector

As was mentioned above T reesei CBHII has beenexpressed in S cerevisiae [8 9] P pastoris [10 11] and Ylipolytica [11] Recombinant CBHII produced in S cerevisiaeshowed an activity of 25 SI UmLminus1 using barely 120573-glucanas substrate [8] while recombinant CBHII produced in Ppastoris GS115 showed an enzyme activity of 584UmLminus1

at 96 h using microcrystalline cellulose PH101 as substrate[10] In P pastoris X-33 and Y lipolytica POld extracellularrecombinant CBHII activities were 025 and 036UmLminus1respectively using phosphoric acid swollen cellulose (PASC)prepared fromAvicel PH-101 as substrate [11]The differencesin promoters secretion signals hosts and enzyme activitysubstrates limit the comparison between these results andthose obtained for recombinant CBHII produced in Wanomalus 54-A However a crude culture broth from Peni-cillium sp containing wild-type cellulase and hemicellulaseenzymes (see Chrysanthemum Wastes Degradation Assay)showed a CBHII activity of 85U Lminus1 using the pNPC sub-strate whichwas similar to that observed for the recombinantCBHII produced in W anomalus 54-A Nevertheless theseresults are lower than those observed for erlenmeyer flaskcultures of Pleurotus ostreatus which showed an activity of4453 plusmn 276ULminus1 using the pNPC substrate [18 19]

SDS-PAGE analysis of extracellular fraction ofW anoma-lus 54-A Celo-32 showed that recombinant CBHII has anapparent molecular weight of about 66 kDa (Figure 1(b))This recombinant CBHII is higher than the wild-type enzymeproduced by T reesei (50ndash58 kDa) [24] However recombi-nant CBHII produced in W anomalus 54-A has a molecularweight similar to that reported for a CBHII produced in Ppastoris X33 and Y lipolytica POld (sim63ndash65 kDa) [11] buthigher than that reported for the enzyme produced in Ppastoris GS115 (sim58 kDa) [10] Differences in the molecularweight between wild-type and the recombinant counterpartcould be associated with the hypermannosylation observed

Enzyme Research 5

2 glu (UL)2 gly (UL)1 glu 1 gly(UL)

2 glu 1 gly(UL)2 glu (Ug)2 gly (Ug)

1 glu 1 gly(Ug)2 glu 1 gly(Ug)

0 24 48 72 96

Induction time (h)

CBH

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tivity

(Ug

dry

wei

ght o

f cel

ls)

0

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CBH

II ac

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(UL

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Figure 2 Effect of the carbon source on the production ofrecombinantT reeseiCBHII Production of recombinantCBHIIwascarried out at 55mLwith 2 (wv) glucose (circle) 2 (wv) glycerol(square) 2 (wv) glucose 1 (wv) glycerol (up triangle) and 1(wv) glucose 1 (wv) glycerol (down triangle) Enzyme activityis reported as U Lminus1 (continuous line) and U gminus1 dry weight of cells(dashed line) Each experiment was done in triplicate

in yeast while the differences in the molecular weight amongthe recombinant CBHII could be associated with severalfactors of the culture conditions that affect the expression ofglycosyltransferases [25]

32 Evaluation of Carbon Sources To improve the produc-tion of recombinant CBHII two glucose and glycerol concen-trations were evaluated First 1wv glucose was evaluatedwhich is lower than the concentration suggested by thevector manufacturer (2wv) We hypothesize that 1wvglucose would be depleted faster than 2wv allowing areduction in the induction time and a longer and higherenzyme production [22] In addition glycerol alone or incombination with glucose was also evaluated as a carbonsource since it was expected that glycerol would be usedfor yeast growth after depletion of glucose increasing theproduction of the recombinant enzyme

Figure 2 shows the results of volumetric activity (U Lminus1)and enzyme activity normalized by biomass (U gminus1 dryweightof cells) A similar activity profile was observed betweenvolumetric and normalized enzyme activities suggesting thatthe production of recombinant CBHII was associated withyeast growth In fact the profile of recombinant CBHIIproduction was similar to that previously reported for theADH2 promoter with a complete induction in the latestationary phase (gt36 h culture) [22]The results showed thatin 2 (wv) glycerol cultures no CBHII activity was detectedsuggesting that the glycerol was used for cell growth or thatthe ADH2 promoter needs to sense the glucose depletion toinduce the gene expression On the other hand lower CBHIIactivity was observed at 2 (wv) glucose 1 (wv) glycerolcultures compared to that obtained with 2 (wv) glucose(119901 lt 0001) Finally the highest CBHII activity was observed

Cel

l den

sity

(gL

)D

issol

ved

oxyg

en (

)

24 48 72 96 1200Culture time (h)

0

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Figure 3 Production of recombinant T reesei CBHII inW anoma-lus 54-A at 24 L scale Recombinant T reesei CBHII was producedat 24 L with 1 (wv) glucose and 1 (wv) glycerol Productionwas followed by 117 h after the final feeding step Dissolved oxygen() cell density (gLminus1) and extracellular enzyme activity (U Lminus1) arereported

at 1 (wv) glucose 1 (wv) glycerol although this wasnot statistically different regarding the levels obtained with2 (wv) glucose (p gt 005) However 1 (wv) glucose 1(wv) glycerol allowed an earlier detection of the enzymeactivity than that observed with 2 (wv) glucose In thissense 1 (wv) glucose 1 (wv) glycerol was selected toproduce recombinant CBHII at 24 L scaleThe reasons of theeffect of glycerol on CBHII activity are unknown and furtherinvestigations should be carried out to clarify this aspectNevertheless it is important to mention that there is a widevariation among yeasts and even strains regarding glycerolmetabolism (ie uptake and catabolism) [26] Although themetabolic pathways and the uptake and regulation (egcatabolic repression) process have been widely described forS cerevisiae limited information is available for other yeasts[26] In this sense the effect of glycerol on the changes ofenzyme activity levels might be associated with the specificglycerol metabolism ofW anomalus 54-A

33 Production of Recombinant CBHII at 24 L Scale Theproduction of recombinant CBHII enzyme at 24 L scalewas carried out for 117 h after the final feeding step (seeMaterials and Methods) Figure 3 shows the behavior ofdissolved oxygen (DO) during the production of recombi-nant CBHII at bioreactor scale which could be associatedwith the consumption of the carbon sources The CBHIIactivity was detected from the first 2 h of culture (012 plusmn008ULminus1)This activity significantly increased after 10 h (26plusmn 009ULminus1) and continuously kept increasing to reach afinal enzyme activity of 145 plusmn 086ULminus1 which was 27-fold higher than that observed at 55mL scale These resultsmight suggest that under controlled culture conditions thestrict repression of the ADH2 promoter might be affected

6 Enzyme Research

0

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re

lativ

e act

ivity

3 4 5 6 7 8 92pH

30∘C

40∘C

50∘C

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Figure 4 Effect of temperature and pH on reaction of recombinantT reeseiCBHIIThe enzyme activity of recombinant T reeseiCBHIIproduced in W anomalus 54-A was evaluated at different pH andtemperatures reactions condition Each experiment was carried outin triplicate Activity is reported as Relative Activity as comparedwith the highest activity obtained in the experiment (40∘C pH 5)

allowing the expression of the recombinant protein even inthe presence of residual glucose

34 Characterization of CBHRecombinant Extract Theeffectof temperature and pHon the enzyme activity of the recombi-nant CBHII produced inW anomalus 54-A was evaluated Inthis work crude extract rather than the purified recombinantCBHII was used for characterization since this crude extractwould be used in the production of bioethanol by thedegradation of Chrysanthemum residues [19] As observedin Figure 4 optimum reaction temperature for recombinantCBHII crude extract was 40∘C (119901 lt 0001) while the highestenzyme activities were observed at pH 50 and 60 plusmn 02 atall evaluated temperatures (119901 lt 0001) On the other handat pH 30 and 80 plusmn 02 a marked decrease in the enzymaticactivity was observed regardless of the reaction temperature

Using microcrystalline cellulose PH101 as substraterecombinant CBHII produced in P pastoris showed anoptimum reaction pH and temperature of 50 plusmn 02 and 50∘Crespectively In addition this recombinant enzyme showeda significant loss of activity at pH of 30 70 and 80 and at30∘C and temperatures above 65∘C [10] Recombinant CBHIIenzyme produced in P pastoris and Y lipolytica showed anoptimum pH between 50 and 60 and at a temperature of60∘C Recombinant CBHII produced in P pastoris and Ylipolytica also showed rapid inactivation at pH and temper-atures above 70 and 60∘C respectively while at temperaturesbelow 40∘C an 80 loss in the enzyme activity was observed[11] In this sense recombinant T reesei CBHII produced inW anomalus 54-A has a reaction pH profile similar to thatreported for recombinant CBHII enzymes expressed in otheryeast Nevertheless it seems that the optimum temperatureof reaction differs among the recombinant CBHII enzymeswhich might be associated with the substrate used for the

enzyme activity assay or the posttranslational modificationscarried out for each host It is noteworthy that recombinantCBHII produced in W anomalus 54-A only showed 30reduction in enzyme activity at 30∘C Functionality at 30∘Cmight allow the use of this enzyme in SSF processes forthe production of bioethanol since this temperature is alsothe optimum growth temperature for S cerevisiae that is thepreferablemicroorganismused during the fermentation stageof bioethanol production [27]

Kinetic parameters for the crude extract were estimatedusing pNPC as substrate (Supplementary Figure 6) Recom-binantT reeseiCBHII produced inWanomalus 54-A showed119870119872andVmax of 273mM(est error 026) and 231 120583Mminminus1

(est error 089) respectivelyThis119870119872value agrees with those

reported for wild-type CBHII enzymes (EC 32191) isolatedfrom different microorganisms which vary between 01 and31mM depending on the source of the enzyme [28] Inthe case of T reesei CBHII enzymes there are no reportsof kinetic parameters using pNPC substrate Neverthelessa wide variation in 119870

119872values is observed (ie between

0041 and 380mM) depending on the substrate used for thisestimation [28]

35 Chrysanthemum Wastes Degradation Assay To evaluatethe effect of recombinant CBHII on cellulose hydrolysisChrysanthemum wastes degradation assay was performedusing three enzyme extracts (1) a crude culture broth fromPenicillium sp containing wild-type cellulase and hemi-cellulase enzymes (Ce-Hem extract) (2) a concentratedrecombinant CBHII extract fromW anomalus 54-A (rCBHIIextract) and (3) a 1 1 Ce-Hem rCBHII extracts mixtureAfter treatment 02 plusmn 002 04 plusmn 001 and 125 plusmn 01 g Lminus1of reducing sugars were observed for Ce-Hem rCBHIIand 1 1 Ce-Hem rCBHII extracts respectively Howeverwhen reducing sugar concentration was normalized by theCBHII units present in each extract at the beginning of theexperiment (Figure 5) it was observed that rCBHII extracthad production of reducing sugars similar to that obtainedwith the Ce-Hem extract (119901 gt 005) Noteworthy theuse of a 1 1 Ce-Hem rCBHII extracts mixture allowed anincrease of about 5-fold in the reducing sugars per CBHIIunit in comparison with the results obtained by using theCe-Hem or concentrated recombinant CBHII extracts alonesuggesting a synergistic effect of the combination of thesetwo enzymatic extracts To understand the reasons of thissynergistic effect we measured the activity of 120573-glucosidase(BG) and endoglucanase (EG) in the extracts (Figure 5)Whereas BG was detected in the three evaluated extracts EGwas only detected in Ce-Hem and Ce-Hem rCBHII extractsNevertheless there was no correlation between BG and EGactivity and the increase in reducing sugars observed withthe Ce-Hem rCBHII extract In this sense we hypothesizethat the synergistic effect observed in the Ce-Hem rCBHIItreatment could be associated with the presence of otherenzymes such as lignin peroxidase xylanase laccase andmanganese peroxidase [29]

Chrysanthemum wastes degradation using enzymaticextracts with native or recombinant cellulases has not been

Enzyme Research 7

Ce-Hem extract rCBHII rCBHIICe-Hem extract

CBHII 85Enzyme activity (UL)

000

005

010

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Redu

cing

suga

rs p

er C

BHII

uni

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115181BGEG

1501819ND

303235241089

lowastlowastlowastlowast


Figure 5 Chrysanthemum wastes degradation assay The effect ofrecombinant CBHII on cellulose hydrolysis was evaluated by usingChrysanthemum wastes Flasks that contained 1 Chrysanthemumwastes were incubated for 20 days at 150 rpm and 30∘C with a crudeculture broth fromPenicillium sp containingwild-type cellulase andhemicellulase enzymes (Ce-Hem extract) concentrated recombi-nant CBHII extract from W anomalus 54-A (rCBHII) and a 1 1Ce-Hem rCBHII extracts mixture Results are reported as g Lminus1 ofreducing sugars per unit of CBHII after subtraction of the resultsobtained with the control cultures lowast lowast lowastlowast = 119901 lt 00001 Thetable shows the enzyme activities of CBHII 120573-glucosidase (BG)and endoglucanase (EG) present in the enzymatic extracts at thebeginning of the assay

previously reported For this lignocellulosic waste degra-dation with P ostreatus showed a production of 96 g Lminus1of reducing sugars [18] The higher production of reducingsugars using P ostreatus could be associated with the pro-duction of several lignocellulolytic enzymes by P ostreatussuch as laccases (up to 6 isoforms) manganese peroxidasescellulases and xylanases [30ndash32] The presence of all theselignocellulolytic enzymes has shown an improvement in thedelignification and reducing sugars production processes [2933] In addition the use of enzymatic extracts with recombi-nant cellulases to degrade lignocellulosic materials has beenpreceded by chemical or physical-chemical delignificationtreatments such as alkaline [10] or steam [34] pretreatmentsThese pretreatments allowed higher reducing sugar levelsthan those reported in the present study (between 12 and20 g Lminus1) showing the importance of lignin degradation toincrease biomass digestibility [29] Nevertheless these resultsshowed that an enzymatic extract of W anomalus 54-Acarrying a recombinant T reesei CBHII allows productionof reducing sugars similar to that of a crude extract fromcellulolytic fungi showing the potential ofW anomalus 54-Aas a host to produce recombinant cellulases

In conclusion we reported the production of therecombinant T reesei CBHII in W anomalus 54-A strainsThe results showed production of recombinant CBHII inW anomalus 54-A with enzyme activity levels of up to145U Lminus1 Recombinant CBHII showed optimum pH andtemperature reaction of 50ndash60 plusmn 02 and 40∘C respectively

which were similar to those reported for other recombinantT reesei CBHII enzymes Km of this recombinant CBHIIwas between the ranges of values reported for other CBHIIenzymes The results showed that an enzymatic extract ofW anomalus 54-A carrying the recombinant T reesei CBHIIallows production of reducing sugars similar to that of a crudeextract from cellulolytic fungi showing the potential of Wanomalus 54-A as a host to produce recombinant cellulasesNoteworthily this is the first report on the use ofW anomalusas a host to produce heterologous proteins Further studiesshould be focused on the consolidation of W anomalus asa platform to produce recombinant proteins such as thedesign of specific expression vectors and the generation ofauxotrophic strains

Additional Points

One-Sentence SummaryThis work represents the first reporton the use of the yeastWickerhamomyces anomalus as a hostto produce heterologous proteins

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors thank Dr Pilar Melendez from PharmacyDepartment at Universidad Nacional de Colombia for thekind donation of theWanomalusA-54 strainDennis J Dıaz-Rincon and Alexander Rodrıguez-Lopez received youngresearcher and doctoral scholarship respectively from Pon-tificia Universidad Javeriana Angela Espejo-Mojica receiveda doctoral scholarship from the Administrative Departmentof Science Technology and Innovation Colciencias Theauthors thankNicolasContreras for his assistance during per-forming experiments This work was supported by PontificiaUniversidad Javeriana [Grant ID 00005578 Produccion deEtanol Mediante la Degradacion de Residuos de CrisantemoEmpleando Enzimas Recombinantes Producidas en Saccha-romyces cerevisiae]

References

[1] L Cuervo J L Folch and R E Quiroz ldquoLignocelulosa comofuente de azucares para la produccion de etanolrdquo BioTecnologıavol 13 pp 11ndash25 2009

[2] V Juturu and J C Wu ldquoMicrobial cellulases Engineering pro-duction and applicationsrdquo Renewable and Sustainable EnergyReviews vol 33 pp 188ndash203 2014

[3] R C Kuhad R Gupta and A Singh ldquoMicrobial cellulases andtheir industrial applicationsrdquoEnzymeResearch vol 2011 ArticleID 280696 11 pages 2011

[4] J Zhou Y-H Wang J Chu L-Z Luo Y-P Zhuang and S-LZhang ldquoOptimization of cellulase mixture for efficient hydrol-ysis of steam-exploded corn stover by statistically designedexperimentsrdquo Bioresource Technology vol 100 no 2 pp 819ndash825 2009

[5] M Tanghe B Danneels A Camattari et al ldquoRecombinant Ex-pression of Trichoderma reesei Cel61A in Pichia pastoris

8 Enzyme Research

Optimizing Yield and N-terminal Processingrdquo MolecularBiotechnology vol 57 no 11-12 pp 1010ndash1017 2015

[6] S H Petersen W H Van Zyl and I S Pretorius ldquoDevelop-ment of a polysaccharide degrading strain of Saccharomycescerevisiaerdquo Biotechnology Techniques vol 12 no 8 pp 615ndash6191998

[7] J H D Van Zyl R Den Haan and W H Van Zyl ldquoOver-expression of native Saccharomyces cerevisiae exocytic SNAREgenes increased heterologous cellulase secretionrdquo AppliedMicrobiology and Biotechnology vol 98 no 12 pp 5567ndash55782014

[8] M Bailey M Siika-aho A Valkeajarvi and M PenttilaldquoHydrolytic properties of two cellulases of Trichoderma reeseiexpressed in yeastrdquoBiotechnology andApplied Biochemistry vol17 no 1 pp 65ndash76 1993

[9] M E Penttila L Andre P Lehtovaara M Bailey T TTeeri and J K C Knowles ldquoEfficient secretion of two fungalcellobiohydrolases by Saccharomyces cerevisiaerdquo Gene vol 63no 1 pp 103ndash112 1988

[10] H Fang and L Xia ldquoHeterologous expression and productionofTrichoderma reesei cellobiohydrolase II in Pichia pastorisand the application in the enzymatic hydrolysis of corn stoverand rice strawrdquo Biomass and Bioenergy vol 78 pp 99ndash109 2015

[11] N Boonvitthya S Bozonnet V Burapatana M J OrsquoDonohueand W Chulalaksananukul ldquoComparison of the heterologousexpression of trichoderma reesei endoglucanase II and cel-lobiohydrolase II in the yeasts pichia pastoris and yarrowialipolyticardquo Molecular Biotechnology vol 54 no 2 pp 158ndash1692013

[12] A L Demain and P Vaishnav ldquoProduction of recombinantproteins by microbes and higher organismsrdquo BiotechnologyAdvances vol 27 no 3 pp 297ndash306 2009

[13] J L Corchero B Gasser D Resina et al ldquoUnconventionalmicrobial systems for the cost-efficient production of high-quality protein therapeuticsrdquo Biotechnology Advances vol 31no 2 pp 140ndash153 2013

[14] V Passoth M Olstorpe and J Schnurer ldquoPast present andfuture research directions with Pichia anomalardquo Antonie vanLeeuwenhoek International Journal of General and MolecularMicrobiology vol 99 no 1 pp 121ndash125 2011

[15] L De Vuyst H Harth S Van Kerrebroeck and F Leroy ldquoYeastdiversity of sourdoughs and associated metabolic propertiesand functionalitiesrdquo International Journal of FoodMicrobiologyvol 239 pp 26ndash34 2016

[16] T N Petersen S Brunak G Von Heijne and H Nielsen ldquoSig-nalP 40 discriminating signal peptides from transmembraneregionsrdquo Nature Methods vol 8 no 10 pp 785-786 2011

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

[18] B Quevedo-Hidalgo P C Narvaez-Rincon A M Pedroza-Rodrıguez and M E Velasquez-Lozano ldquoDegradation ofchrysanthemum (dendranthema grandiflora) wastes by pleuro-tus ostreatus for the production of reducing sugarsrdquo Biotechnol-ogy and Bioprocess Engineering vol 17 no 5 pp 1103ndash1112 2012

[19] B Quevedo-Hidalgo F Monsalve-Marın P C Narvaez-Rincon A M Pedroza-Rodrıguez and M E Velasquez-Lozano ldquoEthanol production by Saccharomyces cerevisiae usinglignocellulosic hydrolysate from Chrysanthemum waste degra-dationrdquo World Journal of Microbiology and Biotechnology vol29 no 3 pp 459ndash466 2013

[20] N Ali Z Ting H Li et al ldquoHeterogeneous Expression andFunctional Characterization of Cellulose-Degrading Enzymesfrom Aspergillus niger for Enzymatic Hydrolysis of AlkaliPretreated Bamboo Biomassrdquo Molecular Biotechnology vol 57no 9 pp 859ndash867 2015

[21] C P Kurtzman ldquoPhylogeny of the ascomycetous yeasts and therenaming of Pichia anomala to Wickerhamomyces anomalusrdquoAntonie van Leeuwenhoek International Journal of General andMolecular Microbiology vol 99 no 1 pp 13ndash23 2011

[22] K M Lee and N A DaSilva ldquoEvaluation of the Saccharomycescerevisiae ADH2 promoter for protein synthesisrdquo Yeast vol 22no 6 pp 431ndash440 2005

[23] V L Price W E Taylor W Clevenger M Worthingtonand E T Young ldquoExpression of heterologous proteins inSaccharomyces cerevisiae using the ADH2 promoterrdquoMethodsin Enzymology vol 185 pp 308ndash318 1990

[24] T T Teeri P Lehtovaara S Kauppinen I Salovuori and JKnowles ldquoHomologous domains in Trichoderma reesei cellu-lolytic enzymes Gene sequence and expression of cellobiohy-drolase IIrdquo Gene vol 51 no 1 pp 43ndash52 1987

[25] B Laukens C De Visscher and N Callewaert ldquoEngineeringyeast for producing human glycoproteins Where are we nowrdquoFuture Microbiology vol 10 no 1 pp 21ndash34 2015

[26] M Klein S Swinnen J MThevelein and E Nevoigt ldquoGlycerolmetabolism and transport in yeast and fungi establishedknowledge and ambiguitiesrdquo Environmental Microbiology vol19 no 3 pp 878ndash893 2017

[27] D Kennes H N Abubackar M Diaz M C Veiga and CKennes ldquoBioethanol production from biomass Carbohydratevs syngas fermentationrdquo Journal of Chemical Technology andBiotechnology vol 91 no 2 pp 304ndash317 2016

[28] A Chang I Schomburg S Placzek et al ldquoBRENDA in 2015Exciting developments in its 25th year of existencerdquo NucleicAcids Research vol 43 no 1 pp D439ndashD446 2015

[29] O Sanchez R Sierra and C J Almeciga-Dıaz ldquoDelignificationProcess of Agro-Industrial Wastes an Alternative to ObtainFermentable Carbohydrates for Producing Fuelrdquo in AlternativeFuel M Manzanera Ed InTech pp 111ndash154 Rijeka 2011

[30] R Castanera G Perez A Omarini et al ldquoTranscriptionaland enzymatic profiling of pleurotus ostreatus laccase genes insubmerged and solid-state fermentation culturesrdquo Applied andEnvironmentalMicrobiology vol 78 no 11 pp 4037ndash4045 2012

[31] J M R da Luz M D Nunes S A Paes D P Torres M DC S da Silva and M C M Kasuya ldquoLignocellulolytic enzymeproduction of Pleurotus ostreatus growth in agroindustrialwastesrdquo Brazilian Journal of Microbiology vol 43 no 4 pp1508ndash1515 2012

[32] O S Isikhuemhen and N A Mikiashvilli ldquoLignocellulolyticenzyme activity substrate utilization and mushroom yield byPleurotus ostreatus cultivated on substrate containing anaerobicdigester solidsrdquo Journal of Industrial Microbiology and Biotech-nology vol 36 no 11 pp 1353ndash1362 2009

[33] J Placido and S Capareda ldquoLigninolytic enzymes a biotechno-logical alternative for bioethanol productionrdquo Bioresources andBioprocessing vol 2 no 23 pp 1ndash12 2015

[34] S P Das A Ghosh A Gupta A Goyal and D Das ldquoLigno-cellulosic fermentation of wild grass employing recombinanthydrolytic enzymes and fermentative microbes with effectivebioethanol recoveryrdquo BioMed Research International vol 2013Article ID 386063 2013

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology

2 Enzyme Research

of cellulose polymer molecules have been recombinantlyexpressed [5] which might allow reducing the produc-tion times and increasing the low proteins concentrationsobtained with filamentous fungi Recombinant expressionmight also facilitate the scaling up of the enzyme productionas well as the implementation of Simultaneous Saccharifica-tion and Fermentation (SSF) and Separate Hydrolysis andFermentation (SHF) processes Saccharomyces cerevisiae hasbeen used for the production of recombinant CBH fromPhanerochaete chrysosporium [6] and Talaromyces emersonii[7] while T reesei CBHII has been expressed in S cerevisiae[8 9] P pastoris [10 11] and Y lipolytica [11]

Conventional microbial systems (eg E coli S cerevisiaeand P pastoris) have allowed the production of a long listof recombinant proteins [12] However there is a growinginterest in the identification of new hosts that allow the pro-duction of high-quality or cost-efficient recombinant proteins[13] Recently a native strain of Wickerhamomyces anomaluswas isolated from sugar cane bagasse at Puerto Lopez MetaColombia (unpublished results) W anomalus is a yeast thathas been isolated from different sources [14] and its reportedapplications include food biopreservation bioremediationand production of phytases and biofuels [14 15] However tothe best of our knowledge there are no reports showing itsuse as host to produce recombinant proteins

In this paper we describe the production of recombinantcellobiohydrolase II (CBHII EC 32191) from T reesei usingthe native strainW anomalus 54-A Furthermore the effectsof pH and temperature on enzyme activity were character-ized as well as the kinetic properties of the enzyme andits application on the hydrolysis of a lignocellulose materialOverall the results showed the potential ofW anomalus 54-A as host to produce recombinant proteins and showed thatrecombinant T reesei CBHII has similar characteristics tothose reported for the wild-type enzyme

2 Materials and Methods

21Microorganism CultureMedia andVectors W anomalus54-A was previously isolated from sugar cane bagasse atPuerto Lopez Meta Colombia Initially the microorgan-ism was identified by carbon assimilation profile using theAPI 20C AUX kit (bioMerieux Marcy lrsquoEtoile France)Further microorganism identification was done by ampli-fication of internal transcribed spacer (ITS) using theprimers ITS1 51015840-TCCGTAGGTGAACCTGCGG-31015840 and ITS451015840-TCCTCCGCTTATTGATATGC-31015840 and Pfu DNA poly-merase (Thermo Fisher Scientific Inc San Jose CA US)undermanufacturer instructions Identity ofWanomalus 54-A was finally confirmed byMALDI-TOF analysis (Unidad deInvestigacion en Proteomica y Micosis Humana PontificiaUniversidad Javeriana)

Plasmid pKS2-ST (Dualsystems Biotech SchlierenSwitzerland) was used as expression vector In this vectorprotein expression is regulated by the alcohol dehydrogenaseII promoter (EC 1111 ADH2) while the secretion signalof the Saccharomyces cerevisiae invertase SUC2 mediatesthe secretion of the recombinant protein Escherichiacoli DH5 was used for cloning purposes which was

cultured in Luria-Bertani (LB) medium supplemented with100mgmLminus1 ampicillin Yeast cultures were performed inYPD supplemented with 500mgmLminus1 geneticin for theselection of clones and 60mgmLminus1 for protein expressioncultures

22 Expression Vector and Recombinant Strains Cellobio-hydrolase II gene (cbhII) from T reesei (GenBank NumberGU7247631) was previously codon-optimized for its expres-sion in S cerevisiae by GeneArt (Thermo Fisher ScientificInc) (unpublished data) The sequence encoding for theCBHII signal peptide was identified by using SignalP [16]and removed from the gene sequence Comparison of thecodon usage tables of S cerevisiae and W anomalus showeda similar profile between both microorganisms (Supplemen-tary Figure 1 in Supplementary Material available online athttpsdoiorg10115520176980565) Two codons (CGC andCGG) which encode Arg (14 residues 3 in CBHII) showedsignificant differences between both microorganisms Never-theless the other two Arg codons have a comparable usagebetween S cerevisiae and W anomalus In this sense thiscodon optimized sequence was used for the CBHII expres-sion in W anomalus 54-A Codon optimized cbhII gene wasinserted downstream of the SUC2 secretion signal in the vec-tor pKS2-ST to produce pKS2-STCBHII (SupplementaryFigure 2)The pKS2-STCBHII vector was used to transformcompetent cells of W anomalus 54-A by electroporationusing the Gene Pulser electroporation system Xcell (Bio-Rad Laboratories) at 1400V and 200Ω The W anomalus54-A clones were selected in YPD medium supplementedwith 500mgmLminus1 geneticin Clones were confirmed by PCRusing the primers 51015840-GAGGAGAGCATAGAAATGGGG-31015840and 51015840-CAGCAGTAGCCATAGCACCA-31015840 which amplify afragment from cbhII gene The PCR-positive clones wereevaluated at 55mL scale according to vector pKS2-ST man-ufacturerrsquos instructions (Dualsystems Biotech) Briefly eachclone was incubated for 10 h in 10mL YPD after which 15mLof fresh YPD medium was added After 14 h incubation30mL of fresh YPD medium was added to reach a finalculture volume of 55mL After 6 h of incubation it wasexpected that the glucose was exhausted and this timewas considered as the beginning of the induction phase1mL aliquots were taken every 24 h for 96 h to measureextracellular enzyme activity and cell density All the assayswere performed in triplicate at 28∘C and 180 rpm Residualglucose quantitation was carried out by DNS method [17]Crude extracellular fractions of the clone with the highestenzyme activity were loaded and processed by SDS-PAGE

23 Evaluation of Carbon Sources The clone that showedthe highest enzyme activity was used to evaluate the effectof carbon source (ie glucose and glycerol) on the enzymeproduction Cultures were carried out in 2 (wv) tryptoneand 1 (wv) yeast extract supplemented with glyceroland glucose in different concentrations 2 (wv) glucose1 (wv) glucose 1 (wv) glycerol 2 (wv) glucose 1(wv) glycerol and 2 (wv) glycerol All cultures weresupplemented with 60mgmLminus1 geneticin

Enzyme Research 3











4 Enzyme Research

8

6

4

2

0

0 20 40 60 80

CBH

II ac

tivity

(UL

)

Induction time (h)

Celo-32Empty vector

(a)

MW

(kD

a)

21

66

3145

97116

200

92 Ｂ0 Ｂ

(b)






Enzyme Research 5




0 24 48 72 96

Induction time (h)

CBH

II ac

tivity

(Ug

dry

wei

ght o

f cel

ls)

0

2

4

6

8

CBH

II ac

tivity

(UL

)

00

01

02





Cel

l den

sity

(gL

)D

issol

ved

oxyg

en (

)

24 48 72 96 1200Culture time (h)

0

5

10

15

20

CBH

II ac

tivity

(UL

)

0

50

100

DO ()





6 Enzyme Research

0

50

100

re

lativ

e act

ivity

3 4 5 6 7 8 92pH

30∘C

40∘C

50∘C

70∘C













Enzyme Research 7



000

005

010

015

Redu

cing

suga

rs p

er C

BHII

uni

t

115181BGEG

1501819ND

303235241089







Additional Points




Acknowledgments


References






8 Enzyme Research






































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Enzyme Research 3











4 Enzyme Research

8

6

4

2

0

0 20 40 60 80

CBH

II ac

tivity

(UL

)

Induction time (h)

Celo-32Empty vector

(a)

MW

(kD

a)

21

66

3145

97116

200

92 Ｂ0 Ｂ

(b)






Enzyme Research 5




0 24 48 72 96

Induction time (h)

CBH

II ac

tivity

(Ug

dry

wei

ght o

f cel

ls)

0

2

4

6

8

CBH

II ac

tivity

(UL

)

00

01

02





Cel

l den

sity

(gL

)D

issol

ved

oxyg

en (

)

24 48 72 96 1200Culture time (h)

0

5

10

15

20

CBH

II ac

tivity

(UL

)

0

50

100

DO ()





6 Enzyme Research

0

50

100

re

lativ

e act

ivity

3 4 5 6 7 8 92pH

30∘C

40∘C

50∘C

70∘C













Enzyme Research 7



000

005

010

015

Redu

cing

suga

rs p

er C

BHII

uni

t

115181BGEG

1501819ND

303235241089







Additional Points




Acknowledgments


References






8 Enzyme Research






































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 Enzyme Research

8

6

4

2

0

0 20 40 60 80

CBH

II ac

tivity

(UL

)

Induction time (h)

Celo-32Empty vector

(a)

MW

(kD

a)

21

66

3145

97116

200

92 Ｂ0 Ｂ

(b)






Enzyme Research 5




0 24 48 72 96

Induction time (h)

CBH

II ac

tivity

(Ug

dry

wei

ght o

f cel

ls)

0

2

4

6

8

CBH

II ac

tivity

(UL

)

00

01

02





Cel

l den

sity

(gL

)D

issol

ved

oxyg

en (

)

24 48 72 96 1200Culture time (h)

0

5

10

15

20

CBH

II ac

tivity

(UL

)

0

50

100

DO ()





6 Enzyme Research

0

50

100

re

lativ

e act

ivity

3 4 5 6 7 8 92pH

30∘C

40∘C

50∘C

70∘C













Enzyme Research 7



000

005

010

015

Redu

cing

suga

rs p

er C

BHII

uni

t

115181BGEG

1501819ND

303235241089







Additional Points




Acknowledgments


References






8 Enzyme Research






































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Enzyme Research 5




0 24 48 72 96

Induction time (h)

CBH

II ac

tivity

(Ug

dry

wei

ght o

f cel

ls)

0

2

4

6

8

CBH

II ac

tivity

(UL

)

00

01

02





Cel

l den

sity

(gL

)D

issol

ved

oxyg

en (

)

24 48 72 96 1200Culture time (h)

0

5

10

15

20

CBH

II ac

tivity

(UL

)

0

50

100

DO ()





6 Enzyme Research

0

50

100

re

lativ

e act

ivity

3 4 5 6 7 8 92pH

30∘C

40∘C

50∘C

70∘C













Enzyme Research 7



000

005

010

015

Redu

cing

suga

rs p

er C

BHII

uni

t

115181BGEG

1501819ND

303235241089







Additional Points




Acknowledgments


References






8 Enzyme Research






































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

6 Enzyme Research

0

50

100

re

lativ

e act

ivity

3 4 5 6 7 8 92pH

30∘C

40∘C

50∘C

70∘C













Enzyme Research 7



000

005

010

015

Redu

cing

suga

rs p

er C

BHII

uni

t

115181BGEG

1501819ND

303235241089







Additional Points




Acknowledgments


References






8 Enzyme Research






































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Enzyme Research 7



000

005

010

015

Redu

cing

suga

rs p

er C

BHII

uni

t

115181BGEG

1501819ND

303235241089







Additional Points




Acknowledgments


References






8 Enzyme Research






































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

8 Enzyme Research






































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

production of recombinant trichoderma reesei

Documents