research article production by tobacco...

Research ArticleProduction by Tobacco Transplastomic Plants of RecombinantFungal and Bacterial Cell-Wall Degrading Enzymes to Be Usedfor Cellulosic Biomass Saccharification

Paolo Longoni12 Sadhu Leelavathi3 Enrico Doria14 Vanga Siva Reddy3 and Rino Cella1

1Dipartimento di Biologia e Biotecnologie Universita di Pavia Via Ferrata 9 27100 Pavia Italy2Dipartimento de Biologie Vegetale Universite de Geneva 30 Quai Ernest Ansermet Sciences III 1211 Geneve Switzerland3Plant Transformation Group International Center for Genetic Engineering and Biotechnology Aruna Asaf Ali MargNew Delhi 110067 India4Centre of Sustainable Livelihood (CSL) Vaal University of Technology Vanderbijlpark 1900 South Africa

Correspondence should be addressed to Vanga Siva Reddy vsreddyicgebresin and Rino Cella rinocellaunipvit

Received 26 December 2014 Revised 6 April 2015 Accepted 9 April 2015

Academic Editor Lei Zhao

Copyright copy 2015 Paolo Longoni et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Biofuels from renewable plant biomass are gainingmomentumdue to climate change related to atmosphericCO2increaseHowever

the production cost of enzymes required for cellulosic biomass saccharification is a major limiting step in this process Low-costproduction of large amounts of recombinant enzymes by transgenic plants was proposed as an alternative to the conventionalmicrobial based fermentation A number of studies have shown that chloroplast-based gene expression offers several advantagesover nuclear transformation due to efficient transcription and translation systems and high copy number of the transgene Inthis study we expressed in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase Leaf extractscontaining the recombinant enzymes showed the ability to degrade various cell-wall components under different conditions singlyand in combinations In addition our group also tested a previously described thermostable xylanase in combination with acellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate Our resultsdemonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugarsfulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry

1 Introduction

Biofuels are currently obtained from edible vegetable prod-ucts (sucrose starch and triglycerides) but ethical consid-erations as well as problems of economic sustainability havestimulated the development of second and third genera-tion biofuels derived from nonedible cellulosic biomass andlipogenic unicellular algae [1 2] The conversion of plantbiomass and cultivation waste (Agri-Waste) into bioethanolis considered a sustainable process as it (1) reduces thedependency on fossil fuels like coal- and petroleum-basedproducts (2) reduces the negative impact on the environ-ment being a carbon-neutral cycle (3) allows us to obtainsecondary byproducts with application in pharmaceuticaland biotechnological industries from the residual biomass

The plant cell wall is a complex structure consisting of amixture of cellulose hemicelluloses and lignin varying fromplant to plant Cellulose is the most diffuse source of reducedcarbon in the world ranking second only to fossil carbon[3] In order to convert plant biomass into biofuels cell-wallmacromolecules must be depolymerized to sugar monomersthat can be fermented to ethanol or other alcohols with ahigher number of carbons through the action of yeast orbacterial strains Alternatively they can be used as growthsubstrate for lipogenic microorganisms to obtain lipid tobe later transformed in biofuel by different treatments Thecurrent technology adopted to degrade cellulose uses highenergy-consuming approaches in order to destroy its stableparacrystalline portion Several fungi and bacteria synthesizeall the enzymes required to degrade cell-wall polysaccharides

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 289759 10 pageshttpdxdoiorg1011552015289759

2 BioMed Research International

to simple sugars or oligosaccharides from which they obtainthe energy to support their growth Some of these microor-ganisms are thermotolerant and possess enzymes active atmedium-high temperatures (60∘C) reviewed in [4 5] A par-ticularly important need is the availability of large amountsof suitable enzyme cocktails for the saccharification of hugeamounts of cellulosic residues and wastes Current estimatessuggest that about 225M tons of cellulosic biomassyear areavailable in EU alone [6] The cost of enzymes used forsaccharification is one of the three crucial parameters for theeconomical sustainability of biofuel production [7 8]

A number of bacterial and fungal strains able to depoly-merize plant cell walls have been described and characterized[9 10] However the expression level of these enzymes bywild-type strains is generally low The recombinant DNAtechnology in combination with improved bioreactors hasbeen shown to increase significantly the production ofmicro-bial enzymes Prokaryotic and eukaryotic expression systemsbased on recombinant DNA approaches have been employedfor the production of proteinsenzymes of commercial inter-est and their advantages and disadvantages evaluated [5 11ndash14] Enzymes used for industrial applications among whichbiofuel production is found are currently produced viamicrobial fermentation even if the process requires highinvestment production andmaintenance costs Several stud-ies show that proteinenzyme production by plant molecularfarming might offer some advantages over microorganismsas plants have both eukaryotic (nuclear) and prokaryotic(chloroplast) expression systems [15ndash18] that can be usedsingly or in combination Transgenic plants were shown to bea valuable system for the production of a variety of antibodiesproteinsenzymes and vaccines [19] A large number ofgenetically modified crops expressing genes encoding insec-ticidal proteins and enzymes conferring resistance to herbi-cides are grown all over the world [20] However the pro-duction of recombinant proteinsenzymes based on nucleartransformation remained a major limitation as the level ofrecombinant proteins accumulation is generally low Con-versely a chloroplast-based expression system offers severaladvantages with respect to the molecular farming notionPlastid genome (plastome) being prokaryotic in origin usesoperons for the expression of multiple foreign genes undera single promoter As the integration of transgene con-structs takes place through homologous recombination thereis a unique transformation event without any positionaleffects contrary to what is observed in the case of nucleartransformation due to random integration of foreign genesinto the nuclear genome Due to independent plastidialtranscription translation and protein folding machineriesrecombinant genes were generally shown to be expressed inchloroplasts at levels higher than that achieved with nuclear-based expression systems [21] In most plant species amongwhich Nicotiana tabacum the plastome is inherited mater-nally thus avoiding transgene dispersion by pollenMoreovertobacco being a nonfood and nonfeed plant is ideal as arecombinant protein expression system since it does not mixwith the food chain a major issue for regulatory clearancesfor commercial activities [22] The low cultivation cost andease of up-scale production of transplastomic plants (plants

with transformed plastid genome) by simply increasing thecultivation area provide additional advantages More than adecade ago Leelavathi et al [15] were the first to demon-strate the feasibility of accumulating a bacterial thermostablexylanase which has several industrial applications includingthe biofuel industry using a chloroplast genetic engineeringapproach Later this approachhas beenused to express a largenumber of cellulolytic enzymes [16 23ndash26] Besides pointingto chloroplast transformation as a promising technology forthe large scale production of recombinant enzymes the studyof Leelavathi et al [15] also showed that the plant producedrecombinant xylanase retained all biochemical functionssimilarly to the native bacterial one It is also noteworthy thatthermostability of recombinant enzymes is a crucial featuresince it allows us to partially overlap the cellulose pretreat-ment process with its digestion

In the present work we expressed in tobacco chloro-plasts five cellulase genes isolated from different microbialorganisms and a polygalacturonase gene from Aspergillusniger Leaf extracts containing the recombinant enzymesweretested for their ability to degrade various cell-wall compo-nents under different conditions singly and in combinationsAlso the previously described thermostable xylanase [15] wasused in combination with cellulases and a polygalacturonaseto study the cumulative effect on the depolymerization ofcomplex plant biomass Our results demonstrate the feasi-bility of converting cell-wall polysaccharides into reducingsugars using a combination of tobacco cell extracts containingenzymes with compatible temperature and pH optima

2 Materials and Methods

21 Chemicals All the reagents and chemicals were pur-chased from Sigma-Aldrich (St Louis MO USA)

22 Construction of Chloroplast Plastid Transformation Vec-tors for the Over Production of Cellulolytic Enzymes in To-bacco The plastid transformation vector pVSR326 (Figure 1GenBank acc number AF527485) was used to clone all genesused in the study pVSR326 vector contains the aadA codingsequence which confers resistance to both spectinomycinand streptomycin under the constitutive 16S rRNA promoterand with the terminator of rbcL [15 21]

The DNA sequences of genes encoding the enzymesused in the present study stored in the GenBank are GH6CHGG 10762 (Cel6 exoglucanse) and gh7 CHGG 08475(Cel7 endoglucanase) GH45 (EndoV endoglucanase)CHGG 08509 of Chaetomium globosum [27] GH 5 (CelK1endoglucanase) (GenBank acc number AAL83749) fromPaenibacillus sp KCTC8848P GH7 CBH-EG Cel3 exo-cellobiohydrolase from Phanerochaete chrysosporium(AAB46373) TF6A (GenBank acc number M73321) Pga2(GenBank acc number XM 001397030) Vlp2 peroxidase(GenBank acc number XM 001220787) For cloning intotransformation vector gene sequences were either amplifiedby polymerase chain reaction (PCR) using the primers indi-cated in Table 1 or got synthesized based on protein sequence

BioMed Research International 3

Table 1

Gene Forward primer (sequence from 51015840-31015840) Reverse primer (sequence from 51015840-31015840)Cel7 TGCTACATCACCCCCTTCAT GTACTTGCGGTGGATGGACTCel6 GATGTGGGCCAACGACTACT GTGGATGGTCAGCTCCTTGTCel3 ATGGCACAGCAGGCAGGTACAC ATAACACTGGCTGTAATACGGATTCCelK1 ATGGCCAGCGTTAAAGGTTATTACC TTCTGCTGCTGCTTTTGCCTGTTCTGCEndoV TACGCCATGGCTCGCTCTACTCCCATTCTTCG AGCTGAGCTCTTAAAGGCATTGCGAGTACCAGTCGPga2 ATGGACAGCTGCACGTTCACC CTAACAAGAGGCCACCGAAGGVlp2 ATGTCGACCGCAACTCGCACTTTC TTAGGCGTTGACGGTCTTGAACACTF6A ATGTCCCCCAGACCTCTTCGC TCAGCTGGCGGCGCAGGTAAG

Alternatively on the basis of the amino acid sequenceavailable for celK1 (GenBank acc number AAL83749) wedesigned synthetic cDNA according to tobacco chloroplastldquocodon usagerdquo (httpwwwkazusaorjpcodon) to optimizesynthesis and accumulation of the relevant enzyme Allsequences were cloned at NcoI and SacI sites of pVSR326(Genbank acc No AF527485) by replacing the uidA (GUS)reporter gene Sequences containing an internalNcoI restric-tion site (ie Pga2) were cloned in two steps In the firststep the C-terminal end of the Pga2 was cloned as NcoI-SacI fragment and then the N-terminal end of the genes wascloned asNcoI-NcoI fragmentThe orientation of the ATG inrelation to the C-terminal part was confirmed by PCR andsequencing All the genes are placed under the psbA generegulatory elements

23 Plant Transformation andMolecular Analysis Transplas-tomic tobacco (Nicotiana tabacum cv Petit Havana) plantswere obtained using the particle delivery method describedearlier [15] Bombarded leaf explants (T0) were regener-ated on selective RMOP medium containing spectinomycin(500mgL) Regenerated green shoots obtained 30 days afterbombardment were grown to maturity to collect seeds (T1)that were germinated on agar plates containing spectino-mycin and streptomycin (500mgL each) In order to obtainhomotransplastomic lines T1 leaf explants were cultured onRMOPmedium containing spectinomycin and streptomycin(500mgL each)This process was repeated up to three times(T3)

Southern blot analysis was used to confirm site-specificintegration of transgenes and homoplasticity of transplas-tomic plants Total genomic DNA was isolated using theTrizol method (Sigma-Aldrich USA) digested with ClaIseparated on 08 agarose gel and blotted onto Nylon mem-branes that after UV irradiation were probed with 32P labeledDNA corresponding to rbcL-accD DNA flanking region andto the coding region of the genes of interest Northern blotanalysis was carried out to confirm efficient transcriptionof all tested genes In both cases standard procedures werefollowed for hybridization and washing [28]

24 Protein Extraction and Enzyme Activity Following apreliminary screening of activity with leaves of different agefully expanded leaves were used to extract the enzymes ofinterest Crude leaf homogenates were used in all cases

in view of developing a simple and cost-effective indus-trial saccharification process A 1 g leaf sample from eachtransplastomic plant was cut into small pieces and ground ina mortar with liquid nitrogen and 3mL of extraction bufferadded to the resulting powder Acetate or phosphate bufferwas used in the 40ndash80 pH range as indicated The planthomogenate was then mixed and centrifuged for 10min at16873timesg and collected the supernatant in a new Eppendorftube In order to eliminate the presence of the endogenoussugars that may subsequently interfere with the reducingsugar assay and to concentrate it the leaf extract was filteredusing Vivaspin 500 (28-9322-18) columns with a cut-offof 3 kDa The concentration of total soluble protein (tsp)was determined using the Bradford reagent (Sigma-AldrichUSA) according to the manufacturerrsquos instructions For allenzyme assays a concentration of 01mgmL of total solubleprotein (tsp) content was used

25 Preparation of the PoplarWood Powder Thepoplar woodsamples used for laboratory analysis are represented frombranches and stem of a poplar clone supplied by the ldquoFrancoAlasia Vivairdquo company Savigliano (Cuneo) Italy Beforeconducting the experiments the wood samples were driedovernight at 40∘C and then cut into small pieces (length 05ndash1 cm width 2-3mm height 1-2mm) using vineyard scissorsWood chips were then ground to fine powder using the millMM301 from Retch at the frequency of 30 vibrationssec for20 seconds repeating each cycle for three times

26 EnzymaticActivityAssays Cellulase activitywas assayedincubating for 60min at different temperatures in 1mL ofthe plant extract (01mgmL tsp) containing 002 g of car-boxymethylcellulose (CMC) or microcrystalline cel-lulose (MCC) xylanase activitywas determined incubatingthe same extractwith 002 g of xylan in the same experimentalconditions used for the previous assay the same procedurewas adopted to test the polygalacturonase activity usingpolygalacturonic acid or apple pectin as substrates In orderto assess the total hydrolytic activity of the leaf extract 002 gof the wood powder was incubated with 1mL of plant extractthe amount of released reducing sugars was determined bydinitrosalicylic acid (DNS) method [29] A fraction of theincubated extract (250120583L) was added to 250120583L of water andto 15mL of DNS reagent in a 2mL test-tube boiled for 10minutes and then cooled down at room temperature Sample


Nt Vlp2

Nt Pga2

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Nt Cel3

Nt CelK1

accDrbcL

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rbcL-accD probe

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Partial cpDNA

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TF6A(072kb)

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Cel7(133kb)

Cel6(145kb)

Cel3(153kb)

CelK1(11 kb)

Vlp2(107 kb)

Vlp2(107 kb)

Figure 1 Portion of the chloroplast transformation vector map containing the gene coding for Vlp2 (pVSR326Vlp2) integration site oftobacco chloroplast DNA (cpDNA) and the same region in transplastomic tobacco plants are shown Also restriction map of vectorscontaining genes coding for other cellulolytic enzymes is shown All the genes coding for cellulolytic enzymes were put under the expressionsignals of rice psbA Lines with double arrow indicate the size of DNA fragments after the restriction digestion with ClaI restriction enzymeDashed arrow indicates the FLK probe (rbcL-aacD flanking region) used to confirm site specific integration of transgenes A possiblemechanism for site-specific integration of aadA and Vlp2 through two homologous recombination events (crossed lines) is also shownSize of the coding region of each gene is shown in brackets

absorbance at 540 nm was recorded against a water-DNSmixture blank A glucose calibration curve (02ndash05mgmL)was used to determine the amount of reducing sugars(mgg of substrate) after the reaction Celk1 cellulase activitywas tested also using a filter paper as a substrate disksof filter paper (5mm of diameter) were incubated at theindicated temperature with 3mL of the plant extract (pH5) containing 01mgmL of protein content The amount ofsugars released was determined by DNS assay after 15 6and 20 hours of incubation A control sample was preparedincubating the paper sheet with the same volume of acetatebuffer (pH 5) Peroxidase activity of VPL2 was determinedspectrophotometrically at 610 nm monitoring the oxidationof phenol red [30]

3 Results

31 Vector Construction for the Transformation of TobaccoSequences encoding cell-wall degrading enzymes derivedfrom different sources were cloned into pVSR326 vector byreplacing the coding region of reporter uidA gene with thesequence of interest (Figure 1) pVSR326 vector integrates thetransgene cassette into the Single Large Copy region betweenrbcL and accD noncoding region in a site specific manner[21] The recombinant gene encoding the enzyme of interestwas placed under the regulation of chloroplast-specific psbAgene promoter and terminator (Figure 1) The native rbcL-accD region was used as flanking regions for a site-specificintegration of transgenes through two possible homologous


Table 2 Activity of various tobacco chloroplast expressed enzymes on carboxymethylcellulose (CMC) and on microcrystalline cellulose(MCC) substrates

Enzyme Activity Umg protein Type Generation of plants used in the studyCMC MCC

Cel63 03 plusmn 002 05 plusmn 003 Exoglucanase T3Cel3 036 plusmn 002 012 plusmn 0005 Endoglucanase T3CelK1 36 plusmn 015 02 plusmn 003 Endoglucanase T3Cel6 016 plusmn 003 04 plusmn 005 Exoglucanase T1Cel7 025 plusmn 0009 021 plusmn 003 Endoglucanase T1

recombination events (Figure 1) The pVSR326 contained aselectable aadA gene conferring resistance to spectinomy-cinstreptomycin The direction and the size of the expectedtranscripts for all the genes are shown in Figure 1

32 Production of Stable Tobacco Transplastomic Plants TheBio-Rad Biolistic PDS-1000He Particle Delivery System wasused to transform tobacco chloroplasts [21] Transplastomicplants were selected under spectinomycin containin medium[15] Out of 20 leaves bombarded with each construct about30ndash45 green shoots were obtained 30 days after bombard-ment on RMOP selection medium In order to obtain homo-transplastomic lines leaf explants from the regenerated plantswere subcultured again on selective RMOP medium Thisprocess was repeated up to three times and the degree ofhomoplasticity was assessed by southern hybridization Oneof cellulase-producing lines T3 lines cel3 turned white andlost its ability to grow autotrophically Interestingly theseplants could be maintained in the greenhouse in a hetero-plasmic state (see Figure S1 in Supplementary Material avail-able online at httpdxdoiorg1011552015289759) Severepleiotropic effects were also observed with plants expressingBgl1C Cel6B Cel9A and Xeg74 genes from Thermobifidafusca [25] and therefore these lines were not further consid-ered Southern blot hybridization was used to prove stableand site specific integration of transgenes and the selectableaadA gene into the tobacco plastid genome Hybridizationwith the flanking region (rbcL-accD probe) has confirmedsite-specific integration of transgenes into the intergenicregion between rbcL and accD genes (Figure 2) Absence ofany band corresponding to the low molecular weight bandobserved in the wild type plants is a clear indication forthe homotransplastomic nature of their plastome The stableintegration of transgenes into plastid genome was furtherconfirmed by reprobing the blots with gene specific codingsequences as probes An expected size band was observedin all the transformed plants (Figure 2) The aadA gene thatconfers resistance against spectinomycin and streptomycinwas used again to test the progeny for stable inheritance ofthe transgenes in the T1 generation All seedlings derivedfrom seeds produced after self-pollinatination are expectedto remain green when germinated on plates containing bothspectinomycin and streptomycin if the progeny inherit theselectable aadA gene [21]When the seeds obtained after self-pollination of T0 generation plants were germinated on theagar plates containing both spectinomycin and streptomycin

all seedlings remained green while the seedlings from thewild type untransformed plants turned white providingevidence for the stable integration and inheritance of thetransgenes by the progeny plants (data not shown) Further-more northern blot analysis confirmed efficient transcriptionof transgenes since transcripts of the expected sizewere foundin all the transplastomic plants analyzed (Figure 3)The inten-sity of the transcript bands suggests efficient transcription oftransgenes under psbA gene regulatory elements in tobaccochloroplasts In some cases in addition to the expected sizeof transcripts additional minor bands of higher molecularweight were observed These might represent transcripts ofthe same transgenes arising from the rbcL gene promoterpresent upstream to the site of transgene integration

33 Expression of Cell-Wall Degrading Enzymes in Chloro-plasts and Their Biochemical Properties In order to assessthe activity of the chloroplast-accumulated enzymes crudeextracts obtained from healthy tobacco plants were testedusing commercially available substrates or raw wood

Among T3 generation plants those producing CelK1showed the highest cellulase activity at 60∘C in a pH rangeof 50ndash60 and using CMC cellulose as a substrate (Table 2)However as shown in Figure 4 the amount of reducingsugars released dropped considerably when the temperaturewas raised to 70∘C Optimal CelK1 enzyme activity wasobserved at pH 60 and 60∘C (Figure 4) As for the NT Vlp2transplastomic plants we failed to detect peroxidase activityin leaf homogenates and therefore this transformant was notfurther considered

The transplastomic Nt Pga2 plant expressing Pga2showed significant pectinase activity when its leaf extractwas tested on apple pectin substrate The most efficient Pga2activity was observed in the 60ndash80 pH range and at a tem-perature ranging between 60∘C and 70∘C in particular thepolygalacturonidase activity was higher at highest temper-ature and basic conditions (Figure 5(a)) The amount ofreducing molecules (galacturonic acid monomers or oli-gogalacturonides) released at 70∘C and pH 80 wasmore thanfour times the amount of those released at 50∘C and pH70 suggesting that the Pga2 is a thermostable enzyme thatretained its activity when produced in tobacco chloroplasts(Figure 5(a)) Even when Pga2 was tested using raw popularwood as a substrate a very high activity was observed at60∘C and pH 80 (Figure 5(b)) On the other hand despitethe efficient transcription no detectable cellulase activity was


rbcL-accD pga2Probe

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cel6Probe

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tf6AProbe

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celK1Probe

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cel3Probe

231

94

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43

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(kb)M 21

Nt Cel3

M 21

rbcL-accD

rbcL-accD

rbcL-accDrbcL-accD

rbcL-accD

Figure 2 Southern blot hybridization to show site-specific integration of introduced transgenes into tobacco plastid genome for therepresentative transplastomic plant The partial coding region of rbcL and accD was used to show the stable and site specific integrationof transgenes Gene specific DNA probe was also used to confirm the stable integration of the transgene Note the lack of any untransformedplastid DNA in the transplastomic lines Molecular marker (M) wild type (1) and transformed (2) plants

observed in the plants transformed with Cel6 Cel7 TF6Aand EndoV genes

34 A Combination of CelK1 and Xylanase (BSX) or Pga2with Similar Thermostable Properties Improves the Depoly-merization of a Complex Cellulosic Biomass To study thedepolymerization of a complex substrate such as poplar woodpowder we tested a combination of enzymes in differenttemperature and pH conditions CelK1 leaf extract was usedin combination with a homogenate obtained from eithera previously described line overexpressing a thermostablexylanase (BSX) [15 18] or a Pga2 transformed line Sincethe final protein concentration in each assay was 01mgmL

the amount of reducing sugars released from poplar woodwhen assayed at pH 7 with a mixture of CelK1 and BSX wassynergistic as compared to the action of each enzyme alone(Figure 6(a)) The same synergistic effect was also observedwhen raw wood powder was exposed to the action of acombination of CelK1 and Pga2 (Figure 6(b)) As comparedto CelK1 alone the amount of reducing sugars releasedincreased by more than twofold when Pga2 was present inthe reaction mixture These results cannot be explained onlyby the fact that the two enzymes use different substrates butrather suggest that the removal of pectin or xylan makescellulose more accessible to Celk1 On the basis of theseencouraging results we tested a mixture of the three enzymes(BSX Pga2 and CelK1) for the ability to release reducing


vlp2 TF6A PGA2 endoV cel6 cel7 celK1 cel3

Wt PT Wt PT Wt PTWt PT Wt PT Wt PT Wt PT Wt PTProbe

16Sr RNA

lowastlowast

lowast

Figure 3 Northern blot analysis showing the expression of transgenes in tobacco chloroplasts RNA isolated from untransformed control(Wt) and plastid transformed (PT) was separated on formaldehyde-agarose gels blotted on to the Hybond-N+ membrane and hybridizedwith gene specific probes For the loading control the same blots were hybridized again with 16S rRNA probe (lower panel) (1) Nt Vlp2(2) Nt TF6A (3) Nt Pga2 (4) Nt EndoV (5) Nt Cel6 (6) Nt Cel7 (7) Nt CelK1 and (8) Nt Cel3 The red asterisks indicate the putativelonger transcript initiated by the upstream rbcL promoter element

00

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30 40 50 60 70

Redu

cing

suga

rs re

leas

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gm

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Temperature (∘C)

pH 4pH 5

pH 6pH 7

Figure 4 Reducing sugar released by the recombinant endoglu-canase Nt CelK1 in different temperature and pH conditions usingCMC as a substrate The final protein concentration in each assaywas 01mgmL The activity values are expressed as mgmL ofreducing sugars assayed by the DNS method

sugars As shown in Figure 7 the best results were obtained at70∘C and pH 8 However since the temperature optimum ofCelK1 is 60∘C it might be advisable to perform the digestionof the biomass in two steps treat the raw wood powderfirst with BSX andor Pga2 at 70∘C and then add Celk1 andcontinue the incubation at 60∘C As far as the temperatureis concerned this is a particular interesting result since atthe industrial process for the production of bioethanol thewoody biomass is subjected to a heat treatment of over 100∘C(steam explosion) before the enzyme addition and thus thepossibility of adding cell-wall degrading enzymes at 70∘C

might effectively contribute to reduce the saccharificationtime and contribute to speed up the industrial process

4 Discussion

Expression of cell-wall degrading enzymes in plants using anuclear-based transformation approach is a major challengeas the cellulolytic enzyme(s) can interact with the plantcell wall and thereby interfere with cell growth and plantdevelopment [31] To prevent potentially harmful conse-quences caused by recombinant cell-wall degrading enzymesa number of strategies were evaluated among which target-ing to subcellular compartments [32] rhizosecretion intohydroponic culture medium [33] and accumulation of afusion storage proteins in seed oil bodies [34] Howeverall these approaches are characterized by a low expressionof recombinant enzymes generally associated with nucleartransformation and expression system Thus chloroplasttransformation was deemed more suitable to obtain a highlevel of accumulation of recombinant proteins Althoughchloroplast transformation offers the possibility of poly-cistronic transcription we chose to express a single enzymeper transplastomic plant for two main reasons First singlecell-wall degrading enzymes find large industrial applicationFor instance cellulases are used in the textile industry (stone-washing) [35] while xylanases are used for pulp whiteningand animal feed processing [36] Moreover the availabilityof a repertoire of single enzymes allows a better formulationof the most suitable cocktail optimal for each lignocellulosicbiomass available (woody biomass grasses wastepaper etc)

Secondly whenever an enzyme cocktail is required theavailability of single enzymes offers the possibility to planthe timely addition of different enzymes For instance theefficiency at which cell-wall cellulose can be digested will


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Figure 5 Activity of the recombinant polygalacturonase Nt Pga2 in different pH and temperature conditions using different substrates(a) apple pectin (Sigma-Aldrich) (b) raw poplar wood The final protein concentration in each assay was 01mgmL The activity values areexpressed as mgmL of reducing sugars

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Figure 6 Activity of recombinant Nt CelK1 in combination with recombinant Nt BSX (a) or recombinant Nt Pga2 (b) on raw poplar woodas substrate The final protein concentration in each assay was 01mgmL The activity is expressed as mgmL of reducing sugars released

be improved if the biomass is pretreated with a polygalac-turonidase before the addition of cellulases In fact a com-bination of CelK1 and PGA2 enzymes showed an additiveeffect on the release of reducing sugars from poplar wood(Figure 6(b)) Interestingly when both CelK1 and PGA2were used together the amount of reducing sugars releasedincreased by more than twofold those suggesting that theremoval of pectin by PGA2 is making cellulose more accessi-ble to CelK1

A third important reason to avoid a simultaneous mul-tiple expression of several genes refers to a possible incom-patibility of accumulation of a given protein with chloroplastphysiology In fact it was observed that plants singly express-ing bgl1C cel6B cel9A and xeg74 genes from T fusca showedsevere pleiotropic effects [25]Therefore the interference of asingle protein with chloroplast biogenesis andor stability ofthe photosynthetic apparatusmight hamper the expression ofthe remaining ones

In the biorefinery process for the production of bioetha-nol a pretreatment of plant biomass is required to make cell-wall polymers more accessible to the enzymes required fortheir deconstruction [37] Although energy-consuming suchpretreatment is necessary to reduce the amount of enzymeswhich represent the most relevant cost of the entire process[38] It is tempting to speculate that plant molecular farmingdue to the ease of large scale production of recombinantenzymes might effectively contribute to reduce the saccha-rification cost

In conclusion this study proves that a combination ofthree enzymes targeting different components of the plant cellwall but having compatible temperature and pH optima notonly improves the saccharification of cellulose present in acomplex plant biomass but also reduces the number of stepsinvolved in the downstream processing Our future endeavorwould include identification of factors involved in the lowor lack of expressionaccumulation of beta-glycosidase (Bgl)


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60∘

70∘

Figure 7 Activity assays of the enzymatic cocktail composed byrecombinant Nt BSX Nt CelK1 and Nt Pga2 in different pH andtemperature conditions Raw popular wood was used as substrateThe activity is expressed as the concentration of reducing sugarreleased

and also identify Bgl genes from other sources having suitablebiochemical properties in order to improve further the cel-lulosic biomass saccharification

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Paolo Longoni and Sadhu Leelavathi contributed equally

Acknowledgments

The authors thank Ranjana Pathak and Abhishek Das fortheir help in southern and northern analyses The authorsthank Professor Felice Cervone for providing thePga2 codingsequence The authors gratefully acknowledge FondazioneBussolera-Branca for generous financial support Also theauthors are thankful to ICGEB and DBT Government ofIndia The authors are indebted to Dr Fabio Pierotti Cei andProfessor Roberto Schmid for their continuous support andinterest in the work

References

[1] A Carroll and C Somerville ldquoCellulosic biofuelsrdquo AnnualReview of Plant Biology vol 60 pp 165ndash182 2009

[2] S A Scott M P Davey J S Dennis et al ldquoBiodiesel from algaechallenges and prospectsrdquo Current Opinion in Biotechnologyvol 21 no 3 pp 277ndash286 2010

[3] L R Lynd M S Laser D Bransby et al ldquoHow biotech cantransformbiofuelsrdquoNature Biotechnology vol 26 no 2 pp 169ndash172 2008

[4] L E Taylor Z Dai S R Decker et al ldquoHeterologous expressionof glycosyl hydrolases in planta a new departure for biofuelsrdquoTrends in Biotechnology vol 26 no 8 pp 413ndash424 2008

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

[6] S Searle and C Malins White PapermdashAvailability of CellulosicResidues and Wastes in the EU The International Council onClean Transportation (ICCT) Washington DC USA 2013httpwwwtheicctorg

[7] R Wooley M Ruth J Sheehan K Ibsen H Majdeski and AGalvez ldquoLignocellulosic biomass to ethanol process design andeconomics utilizing co-current dilute acid prehydrolysis andenzymatic hydrolysis current and futuristic scenariosrdquo TechRep NRELTP-580-26157 1999

[8] V Lunin ldquoNew cellulase identification method holds promisefor lower-cost biofuelsrdquo Tech Rep NRELFS-2700-58228National Renewable Energy Laboratory 2013

[9] D E Koeck A Pechtl V V Zverlov and W H SchwarzldquoGenomics of cellulolytic bacteriardquo Current Opinion in Biotech-nology vol 29 pp 171ndash183 2014

[10] Z Zhao H Liu C Wang and J-R Xu ldquoComparative analysisof fungal genomes reveals different plant cell wall degradingcapacity in fungirdquoBMCGenomics vol 14 no 1 article 274 2013

[11] J A Fernandez-Robledo and G R Vasta ldquoProduction ofrecombinant proteins from protozoan parasitesrdquo Trends inParasitology vol 26 no 5 pp 244ndash254 2010

[12] D L Hacker M de Jesus and F M Wurm ldquo25 years ofrecombinant proteins from reactor-grown cellsmdashwhere do wego from hererdquo Biotechnology Advances vol 27 no 6 pp 1023ndash1027 2009

[13] G Potvin and Z Zhang ldquoStrategies for high-level recombinantprotein expression in transgenic microalgae a reviewrdquo Biotech-nology Advances vol 28 no 6 pp 910ndash918 2010

[14] R Surzycki K Greenham K Kitayama et al ldquoFactors effectingexpression of vaccines in microalgaerdquo Biologicals vol 37 no 3pp 133ndash138 2009

[15] S Leelavathi N Gupta S Maiti A Ghosh and V S ReddyldquoOverproduction of an alkali- and thermo-stable xylanase intobacco chloroplasts and efficient recovery of the enzymerdquoMolecular Breeding vol 11 no 1 pp 59ndash67 2003

[16] D Verma A Kanagaraj S Jin N D Singh P E Kolattukudyand H Daniell ldquoChloroplast-derived enzyme cocktails hydrol-yse lignocellulosic biomass and release fermentable sugarsrdquoPlant Biotechnology Journal vol 8 no 3 pp 332ndash350 2010

[17] N Scotti M M Rigano and T Cardi ldquoProduction of foreignproteins using plastid transformationrdquo Biotechnology Advancesvol 30 no 2 pp 387ndash397 2012

[18] L Pantaleoni P Longoni L Ferroni et al ldquoChloroplast molec-ular farming efficient production of a thermostablexylanase byNicotiana tabacum plants and long-term conservation of therecombinant enzymerdquo Protoplasma vol 251 pp 639ndash648 2014

[19] H Daniell N D Singh H Mason and S J Streatfield ldquoPlant-made vaccine antigens and biopharmaceuticalsrdquoTrends in PlantScience vol 14 no 12 pp 669ndash679 2009

[20] J Clive ldquoGlobal status of commercialized biotechGM crops2013rdquo ISAAA Brief no 46 International Service for the Acqui-sition of Agri-biotech Applications (ISAAA) Ithaca NY USA2013


[21] V S Reddy S Leelavathi A Selvapandiyan et al ldquoAnalysis ofchloroplast transformed tobacco plants with cry1Ia5 under ricepsbA transcriptional elements reveal high level expression of Bttoxin without imposing yield penalty and stable inheritance oftransplastomerdquo Molecular Breeding vol 9 no 4 pp 259ndash2692002

[22] J Watson V Koya S H Leppla and H Daniell ldquoExpression ofBacillus anthracis protective antigen in transgenic chloroplastsof tobacco a non-foodfeed croprdquoVaccine vol 22 no 31-32 pp4374ndash4384 2004

[23] L-X Yu B N Gray C J Rutzke L P Walker D B Wilsonand M R Hanson ldquoExpression of thermostable microbial cel-lulases in the chloroplasts of nicotine-free tobaccordquo Journal ofBiotechnology vol 131 no 3 pp 362ndash369 2007

[24] S Jung S Kim H Bae H-S Lim and H-J Bae ldquoExpressionof thermostable bacterial beta-glucosidase (BglB) in transgenictobacco plantsrdquo Bioresource technology vol 101 no 18 pp 7155ndash7161 2010

[25] K Petersen and R Bock ldquoHigh-level expression of a suite ofthermostable cell wall-degrading enzymes from the chloroplastgenomerdquo Plant Molecular Biology vol 76 no 3ndash5 pp 311ndash3212011

[26] P Agrawal D Verma and H Daniell ldquoExpression of Tri-choderma reesei 120573-mannanase in tobacco chloroplasts and itsutilization in lignocellulosic woody biomass hydrolysisrdquo PLoSONE vol 6 no 12 Article ID e29302 2011

[27] P Longoni M Rodolfi L Pantaleoni et al ldquoFunctional analysisof the degradation of cellulosic substrates by aChaetomium glo-bosum endophytic isolaterdquo Applied and Environmental Microbi-ology vol 78 no 10 pp 3693ndash3705 2012

[28] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratoryManual Cold SpringHarbor Laboratory Press ColdSpring Harbor NY USA 1989

[29] T K Ghose ldquoMeasurement of cellulase activitiesrdquo Pure andApplied Chemistry vol 59 no 2 1987

[30] A B Orth D J Royse and M Tien ldquoUbiquity of lignin-degrading peroxidases among various wood-degrading fungirdquoApplied and Environmental Microbiology vol 59 no 12 pp4017ndash4023 1993

[31] K Herbers I Wilke and U A Sonnewald ldquoA thermostablexylanase from Clostridium thermocellum expressed at highlevels in the apoplast of transgenic tobacco has no detrimentaleffects and is easily purifiedrdquo Nature Biotechnology vol 13 no1 pp 63ndash66 1995

[32] K Herbers H J Flint and U Sonnewald ldquoApoplastic expres-sion of the xylanase and 120573(1-31-4) glucanase domains of thexyn D gene from Ruminococcus flavefaciens leads to functionalpolypeptides in transgenic tobacco plantsrdquoMolecular Breedingvol 2 no 1 pp 81ndash87 1996

[33] N V Borisjuk L G Borisjuk S Logendra F Petersen Y Glebaand I Raskin ldquoProduction of recombinant proteins in plantroot exudatesrdquoNature Biotechnology vol 17 no 5 pp 466ndash4691999

[34] J-H Liu L B Selinger K-J Cheng K A Beauchemin andM M Moloney ldquoPlant seed oil-bodies as an immobilizationmatrix for a recombinant xylanase from the rumen fungusNeo-callimastix patriciarumrdquo Molecular Breeding vol 3 no 6 pp463ndash470 1997

[35] A Miettinen-Oinonen and P Suominen ldquoEnhanced produc-tion of Trichoderma reesei endoglucanases and use of the newcellulase preparations in producing the stonewashed effect on

denim fabricrdquoApplied and Environmental Microbiology vol 68no 8 pp 3956ndash3964 2002

[36] A D Harris and C Ramalingam ldquoXylanases and its applicationin food industry A reviewrdquo Journal of Experimental Sciencesvol 1 pp 1ndash11 2010

[37] M Sticklen ldquoPlant genetic engineering to improve biomasscharacteristics for biofuelsrdquo Current Opinion in Biotechnologyvol 17 no 3 pp 315ndash319 2006

[38] C C Geddes I U Nieves and L O Ingram ldquoAdvances inethanol productionrdquo Current Opinion in Biotechnology vol 22no 3 pp 312ndash319 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


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


to simple sugars or oligosaccharides from which they obtainthe energy to support their growth Some of these microor-ganisms are thermotolerant and possess enzymes active atmedium-high temperatures (60∘C) reviewed in [4 5] A par-ticularly important need is the availability of large amountsof suitable enzyme cocktails for the saccharification of hugeamounts of cellulosic residues and wastes Current estimatessuggest that about 225M tons of cellulosic biomassyear areavailable in EU alone [6] The cost of enzymes used forsaccharification is one of the three crucial parameters for theeconomical sustainability of biofuel production [7 8]

A number of bacterial and fungal strains able to depoly-merize plant cell walls have been described and characterized[9 10] However the expression level of these enzymes bywild-type strains is generally low The recombinant DNAtechnology in combination with improved bioreactors hasbeen shown to increase significantly the production ofmicro-bial enzymes Prokaryotic and eukaryotic expression systemsbased on recombinant DNA approaches have been employedfor the production of proteinsenzymes of commercial inter-est and their advantages and disadvantages evaluated [5 11ndash14] Enzymes used for industrial applications among whichbiofuel production is found are currently produced viamicrobial fermentation even if the process requires highinvestment production andmaintenance costs Several stud-ies show that proteinenzyme production by plant molecularfarming might offer some advantages over microorganismsas plants have both eukaryotic (nuclear) and prokaryotic(chloroplast) expression systems [15ndash18] that can be usedsingly or in combination Transgenic plants were shown to bea valuable system for the production of a variety of antibodiesproteinsenzymes and vaccines [19] A large number ofgenetically modified crops expressing genes encoding insec-ticidal proteins and enzymes conferring resistance to herbi-cides are grown all over the world [20] However the pro-duction of recombinant proteinsenzymes based on nucleartransformation remained a major limitation as the level ofrecombinant proteins accumulation is generally low Con-versely a chloroplast-based expression system offers severaladvantages with respect to the molecular farming notionPlastid genome (plastome) being prokaryotic in origin usesoperons for the expression of multiple foreign genes undera single promoter As the integration of transgene con-structs takes place through homologous recombination thereis a unique transformation event without any positionaleffects contrary to what is observed in the case of nucleartransformation due to random integration of foreign genesinto the nuclear genome Due to independent plastidialtranscription translation and protein folding machineriesrecombinant genes were generally shown to be expressed inchloroplasts at levels higher than that achieved with nuclear-based expression systems [21] In most plant species amongwhich Nicotiana tabacum the plastome is inherited mater-nally thus avoiding transgene dispersion by pollenMoreovertobacco being a nonfood and nonfeed plant is ideal as arecombinant protein expression system since it does not mixwith the food chain a major issue for regulatory clearancesfor commercial activities [22] The low cultivation cost andease of up-scale production of transplastomic plants (plants

with transformed plastid genome) by simply increasing thecultivation area provide additional advantages More than adecade ago Leelavathi et al [15] were the first to demon-strate the feasibility of accumulating a bacterial thermostablexylanase which has several industrial applications includingthe biofuel industry using a chloroplast genetic engineeringapproach Later this approachhas beenused to express a largenumber of cellulolytic enzymes [16 23ndash26] Besides pointingto chloroplast transformation as a promising technology forthe large scale production of recombinant enzymes the studyof Leelavathi et al [15] also showed that the plant producedrecombinant xylanase retained all biochemical functionssimilarly to the native bacterial one It is also noteworthy thatthermostability of recombinant enzymes is a crucial featuresince it allows us to partially overlap the cellulose pretreat-ment process with its digestion

In the present work we expressed in tobacco chloro-plasts five cellulase genes isolated from different microbialorganisms and a polygalacturonase gene from Aspergillusniger Leaf extracts containing the recombinant enzymesweretested for their ability to degrade various cell-wall compo-nents under different conditions singly and in combinationsAlso the previously described thermostable xylanase [15] wasused in combination with cellulases and a polygalacturonaseto study the cumulative effect on the depolymerization ofcomplex plant biomass Our results demonstrate the feasi-bility of converting cell-wall polysaccharides into reducingsugars using a combination of tobacco cell extracts containingenzymes with compatible temperature and pH optima

2 Materials and Methods

21 Chemicals All the reagents and chemicals were pur-chased from Sigma-Aldrich (St Louis MO USA)

22 Construction of Chloroplast Plastid Transformation Vec-tors for the Over Production of Cellulolytic Enzymes in To-bacco The plastid transformation vector pVSR326 (Figure 1GenBank acc number AF527485) was used to clone all genesused in the study pVSR326 vector contains the aadA codingsequence which confers resistance to both spectinomycinand streptomycin under the constitutive 16S rRNA promoterand with the terminator of rbcL [15 21]

The DNA sequences of genes encoding the enzymesused in the present study stored in the GenBank are GH6CHGG 10762 (Cel6 exoglucanse) and gh7 CHGG 08475(Cel7 endoglucanase) GH45 (EndoV endoglucanase)CHGG 08509 of Chaetomium globosum [27] GH 5 (CelK1endoglucanase) (GenBank acc number AAL83749) fromPaenibacillus sp KCTC8848P GH7 CBH-EG Cel3 exo-cellobiohydrolase from Phanerochaete chrysosporium(AAB46373) TF6A (GenBank acc number M73321) Pga2(GenBank acc number XM 001397030) Vlp2 peroxidase(GenBank acc number XM 001220787) For cloning intotransformation vector gene sequences were either amplifiedby polymerase chain reaction (PCR) using the primers indi-cated in Table 1 or got synthesized based on protein sequence


Table 1










Nt Vlp2

Nt Pga2

Nt TF6A

Nt EndoV

Nt Cel7

Nt Cel6

Nt Cel3

Nt CelK1

accDrbcL

rrn

aadA

psbAP

C

C

C

C

rbcL-accD probe

aadA

C C

C C

C C

C C

C C

C C

C C

C

pVSR326Vlp2

Partial cpDNA

347kb

657kb

302kb 320kb

651kb

653kb

695kb

683 kb

660 kb

703 kb

Pga2(101 kb)

TF6A(072kb)

EndoV(103 kb)

Cel7(133kb)

Cel6(145kb)

Cel3(153kb)

CelK1(11 kb)

Vlp2(107 kb)

Vlp2(107 kb)



3 Results













rbcL-accD pga2Probe

231

94

65

43

2320

(kb) M 21

Nt Pga2

M 21 M 21

Nt EndoV

M 21

endoVProbe

231

94

65

43

2320

(kb)

cel6Probe

231

94

65

43

2320

(kb) M 21

Nt Cel6

M 21

tf6AProbe

231

94

65

43

2320

(kb) M 21

Nt TF6A

M 21

celK1Probe

231

94

65

43

2320

(kb)M 21

Nt CelK1

M 21

cel3Probe

231

94

65

43

2320

(kb)M 21

Nt Cel3

M 21

rbcL-accD

rbcL-accD

rbcL-accDrbcL-accD

rbcL-accD








16Sr RNA

lowastlowast

lowast


00

01

02

03

04

05

06

07

30 40 50 60 70

Redu

cing

suga

rs re

leas

ed (m

gm

L)

Temperature (∘C)

pH 4pH 5

pH 6pH 7




4 Discussion




000

050

100

150

200

250

300

350Re

duci

ng su

gars

rele

ased

(m

gm

L)


50∘

60∘

70∘

(a)


Redu

cing

suga

rs re

leas

ed

(mg

mL)

080

070

060

050

040

030

020

010

000

50∘

60∘

(b)


000

020

040

060

080

100

120

Redu

cing

suga

rs re

leas

ed (m

gm

L)


(a)

0

01

02

03

04

05

06

07

08

09

Redu

cing

suga

rs re

leas

ed (m

gm

L)


(b)







10

09

08

07

06

05

04

03

02

01

00

Redu

cing

suga

rs re

leas

ed (m

gm

L)


50∘

60∘

70∘







Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 1










Nt Vlp2

Nt Pga2

Nt TF6A

Nt EndoV

Nt Cel7

Nt Cel6

Nt Cel3

Nt CelK1

accDrbcL

rrn

aadA

psbAP

C

C

C

C

rbcL-accD probe

aadA

C C

C C

C C

C C

C C

C C

C C

C

pVSR326Vlp2

Partial cpDNA

347kb

657kb

302kb 320kb

651kb

653kb

695kb

683 kb

660 kb

703 kb

Pga2(101 kb)

TF6A(072kb)

EndoV(103 kb)

Cel7(133kb)

Cel6(145kb)

Cel3(153kb)

CelK1(11 kb)

Vlp2(107 kb)

Vlp2(107 kb)



3 Results













rbcL-accD pga2Probe

231

94

65

43

2320

(kb) M 21

Nt Pga2

M 21 M 21

Nt EndoV

M 21

endoVProbe

231

94

65

43

2320

(kb)

cel6Probe

231

94

65

43

2320

(kb) M 21

Nt Cel6

M 21

tf6AProbe

231

94

65

43

2320

(kb) M 21

Nt TF6A

M 21

celK1Probe

231

94

65

43

2320

(kb)M 21

Nt CelK1

M 21

cel3Probe

231

94

65

43

2320

(kb)M 21

Nt Cel3

M 21

rbcL-accD

rbcL-accD

rbcL-accDrbcL-accD

rbcL-accD








16Sr RNA

lowastlowast

lowast


00

01

02

03

04

05

06

07

30 40 50 60 70

Redu

cing

suga

rs re

leas

ed (m

gm

L)

Temperature (∘C)

pH 4pH 5

pH 6pH 7




4 Discussion




000

050

100

150

200

250

300

350Re

duci

ng su

gars

rele

ased

(m

gm

L)


50∘

60∘

70∘

(a)


Redu

cing

suga

rs re

leas

ed

(mg

mL)

080

070

060

050

040

030

020

010

000

50∘

60∘

(b)


000

020

040

060

080

100

120

Redu

cing

suga

rs re

leas

ed (m

gm

L)


(a)

0

01

02

03

04

05

06

07

08

09

Redu

cing

suga

rs re

leas

ed (m

gm

L)


(b)







10

09

08

07

06

05

04

03

02

01

00

Redu

cing

suga

rs re

leas

ed (m

gm

L)


50∘

60∘

70∘







Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Nt Vlp2

Nt Pga2

Nt TF6A

Nt EndoV

Nt Cel7

Nt Cel6

Nt Cel3

Nt CelK1

accDrbcL

rrn

aadA

psbAP

C

C

C

C

rbcL-accD probe

aadA

C C

C C

C C

C C

C C

C C

C C

C

pVSR326Vlp2

Partial cpDNA

347kb

657kb

302kb 320kb

651kb

653kb

695kb

683 kb

660 kb

703 kb

Pga2(101 kb)

TF6A(072kb)

EndoV(103 kb)

Cel7(133kb)

Cel6(145kb)

Cel3(153kb)

CelK1(11 kb)

Vlp2(107 kb)

Vlp2(107 kb)



3 Results













rbcL-accD pga2Probe

231

94

65

43

2320

(kb) M 21

Nt Pga2

M 21 M 21

Nt EndoV

M 21

endoVProbe

231

94

65

43

2320

(kb)

cel6Probe

231

94

65

43

2320

(kb) M 21

Nt Cel6

M 21

tf6AProbe

231

94

65

43

2320

(kb) M 21

Nt TF6A

M 21

celK1Probe

231

94

65

43

2320

(kb)M 21

Nt CelK1

M 21

cel3Probe

231

94

65

43

2320

(kb)M 21

Nt Cel3

M 21

rbcL-accD

rbcL-accD

rbcL-accDrbcL-accD

rbcL-accD








16Sr RNA

lowastlowast

lowast


00

01

02

03

04

05

06

07

30 40 50 60 70

Redu

cing

suga

rs re

leas

ed (m

gm

L)

Temperature (∘C)

pH 4pH 5

pH 6pH 7




4 Discussion




000

050

100

150

200

250

300

350Re

duci

ng su

gars

rele

ased

(m

gm

L)


50∘

60∘

70∘

(a)


Redu

cing

suga

rs re

leas

ed

(mg

mL)

080

070

060

050

040

030

020

010

000

50∘

60∘

(b)


000

020

040

060

080

100

120

Redu

cing

suga

rs re

leas

ed (m

gm

L)


(a)

0

01

02

03

04

05

06

07

08

09

Redu

cing

suga

rs re

leas

ed (m

gm

L)


(b)







10

09

08

07

06

05

04

03

02

01

00

Redu

cing

suga

rs re

leas

ed (m

gm

L)


50∘

60∘

70∘







Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












rbcL-accD pga2Probe

231

94

65

43

2320

(kb) M 21

Nt Pga2

M 21 M 21

Nt EndoV

M 21

endoVProbe

231

94

65

43

2320

(kb)

cel6Probe

231

94

65

43

2320

(kb) M 21

Nt Cel6

M 21

tf6AProbe

231

94

65

43

2320

(kb) M 21

Nt TF6A

M 21

celK1Probe

231

94

65

43

2320

(kb)M 21

Nt CelK1

M 21

cel3Probe

231

94

65

43

2320

(kb)M 21

Nt Cel3

M 21

rbcL-accD

rbcL-accD

rbcL-accDrbcL-accD

rbcL-accD








16Sr RNA

lowastlowast

lowast


00

01

02

03

04

05

06

07

30 40 50 60 70

Redu

cing

suga

rs re

leas

ed (m

gm

L)

Temperature (∘C)

pH 4pH 5

pH 6pH 7




4 Discussion




000

050

100

150

200

250

300

350Re

duci

ng su

gars

rele

ased

(m

gm

L)


50∘

60∘

70∘

(a)


Redu

cing

suga

rs re

leas

ed

(mg

mL)

080

070

060

050

040

030

020

010

000

50∘

60∘

(b)


000

020

040

060

080

100

120

Redu

cing

suga

rs re

leas

ed (m

gm

L)


(a)

0

01

02

03

04

05

06

07

08

09

Redu

cing

suga

rs re

leas

ed (m

gm

L)


(b)







10

09

08

07

06

05

04

03

02

01

00

Redu

cing

suga

rs re

leas

ed (m

gm

L)


50∘

60∘

70∘







Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




16Sr RNA

lowastlowast

lowast


00

01

02

03

04

05

06

07

30 40 50 60 70

Redu

cing

suga

rs re

leas

ed (m

gm

L)

Temperature (∘C)

pH 4pH 5

pH 6pH 7




4 Discussion




000

050

100

150

200

250

300

350Re

duci

ng su

gars

rele

ased

(m

gm

L)


50∘

60∘

70∘

(a)


Redu

cing

suga

rs re

leas

ed

(mg

mL)

080

070

060

050

040

030

020

010

000

50∘

60∘

(b)


000

020

040

060

080

100

120

Redu

cing

suga

rs re

leas

ed (m

gm

L)


(a)

0

01

02

03

04

05

06

07

08

09

Redu

cing

suga

rs re

leas

ed (m

gm

L)


(b)







10

09

08

07

06

05

04

03

02

01

00

Redu

cing

suga

rs re

leas

ed (m

gm

L)


50∘

60∘

70∘







Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


000

050

100

150

200

250

300

350Re

duci

ng su

gars

rele

ased

(m

gm

L)


50∘

60∘

70∘

(a)


Redu

cing

suga

rs re

leas

ed

(mg

mL)

080

070

060

050

040

030

020

010

000

50∘

60∘

(b)


000

020

040

060

080

100

120

Redu

cing

suga

rs re

leas

ed (m

gm

L)


(a)

0

01

02

03

04

05

06

07

08

09

Redu

cing

suga

rs re

leas

ed (m

gm

L)


(b)







10

09

08

07

06

05

04

03

02

01

00

Redu

cing

suga

rs re

leas

ed (m

gm

L)


50∘

60∘

70∘







Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


10

09

08

07

06

05

04

03

02

01

00

Redu

cing

suga

rs re

leas

ed (m

gm

L)


50∘

60∘

70∘







Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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