Structural characterization of a new dextran with a low degree of branching produced by Leuconostoc mesenteroides FT045B dextransucrase

ContentslistsavailableatSciVerseScienceDirect

Carbohydrate

Polymers

jo u r n al h om ep a ge : w w w . e l s e v i e r . c o m / l o c a t e / c a r b p o l

Structural

characterization

of

a

new

dextran

with

a

low

degree

of

branching

produced

by

Leuconostoc

mesenteroides

FT045B

dextransucrase

Mary

Helen

Palmuti

Braga

Vettori

_,

_Sandra

_Mara

_Martins

_Franchetti

_,

_Jonas

_Contiero

a_{Unesp–Univ.EstadualPaulista,BiologicalSciencesInstitute,DepartmentofBiochemistryandMicrobiology,LaboratoryofIndustrialMicrobiology,24AAve.,BelaVista,IBRC,Rio}

Claro,SP,CEP13506-900,Brazil

b_{Unesp–Univ.EstadualPaulista,BiologicalSciencesInstitute,DepartmentofBiochemistryandMicrobiology,LaboratoryofPolymerBiotreatments,24AAve.,BelaVista,IBRC,Rio}

Claro,SP,CEP13506-900,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received11October2011

Receivedinrevisedform14February2012 Accepted18February2012

Available online 25 February 2012

Keywords:

LeuconostocmesenteroidesFT045B dextransucrase

Dextran

Structuralcharacterization ComparisonwithcommercialB-512F dextran

a

b

s

t

r

a

c

t

This paper offers thephysical and chemical characterizationof a new dextran producedby

Leu-conostocmesenteroidesFT045B.ThechemicalstructurewasdeterminedbyFourierTransformInfrared

spectroscopyand1_{HNuclearMagneticResonancespectroscopy.Thedextranwashydrolyzedby}

endo-dextranase;theproductswereanalyzedusingthinlayerchromatographyandcomparedwiththoseof

commercialB-512Fdextran.Thenumber-averagemolecularweightanddegreeofpolymerizationofthe

FT045Bdextranweredeterminedbythemeasurementofthereducingvalueusingthecopper

bicinchon-inatemethodandthemeasurementoftotalcarbohydrateusingthephenol–sulfuricacidmethod.The

datarevealedthatthestructureofthedextransynthesizedbyFT045Bdextransucraseiscomposedof

d_{-glucoseresidues,containing97.9%␣-(1,6)linkagesinthemainchainsand2.1%␣-(1,3)branchlinkages}

comparedwiththecommercialB-512Fdextran,whichhas95%␣-(1,6)linkagesinthemainchainsand

5%␣-(1,3)branchlinkages.

© 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Dextran is an extracellular bacterial homopolysaccharide (Sidebotham, 1974; Monsan et al., 2001), _{the main chain of}

whichiscomposedofcontiguous␣-(1,6)-linkedd_{-glucopyranose} residuesand branches, stemmingmainlyside-chains of ␣-(1,6)

glucose units attached by ␣-(1,3) branch linkages to ␣

-(1,6)-linkedchains(Buchholz&Monsan,2001;Dols,Remaud-Simeon, Willemot,Vignon,&Monsan,1998;Naessens,Cerdobbel,Soetaert, &Vandamme,2005;Robyt,1995;Seymour&Knapp,1980).Some dextranshave ␣-(1,2)or ␣-(1,4)branchlinkages, dependingon

thebacterialsource.Theexactstructureofeachtypeofdextran dependsonthedegreeofbranching,involving␣-(1,2),␣-(1,3)and ␣-(1,4)linkages(Seymour&Knapp,1980),whichdependsonthe

specificmicrobialstrainand,hence,onthespecificityof dextran-sucrase(s)EC.2.4.1.5involved(Jeaneset al.,1954).Dextranscan beproducedbygrowingvariousbacterialstrainsofLeuconostoc mesenteroidesandStreptococcusspeciesonamediumcontaining

Abbreviations: DPn,numberaveragedegreeofpolymerization;FTIR,Fourier TransformInfrared;1_HNMR,1_{HNuclearMagneticResonance;MWn,} number-averagemolecularweight;NCBI,NationalCenterforBiotechnologyInformation; Pyr/Ac,Pyridine/Acetate;TFA,trifluoroaceticacid;TLC,thinlayerchromatography.

∗Correspondingauthor.Tel.:+551935264180;fax:+551935264176.

E-mailaddresses:marypalmuti@bol.com.br(M.H.P.B.Vettori),

samaramf@rc.unesp.br(S.M.M.Franchetti),jconti@rc.unesp.br(J.Contiero).

sucrose(Kim,Robyt,Lee,Lee,&Kim,2003).Dextranproductionis influencedbyanumberoffactors:physicalandchemicalproperties (Jeanes,1965),suchassolubility,viscosity,specificopticalrotation andthecontentofnitrogen,phosphorusandashinthemedium, whichfurtherdependsonthespecificmicroorganismsinvolved (Jeanesetal.,1954).Asingleenzymecancatalyzethesynthesisof severaltypesofdextranlinkages,therebypermittingthe forma-tionofabranchedpolymer(Neely&Nott,1962;Smith,Sahnley,& Goodman,1994).Ontheotherhand,certainbacterialstrainshave beenshowntoproducedextransofdifferentstructures,whichhave beenattributedtotheexcretionofdifferentdextransucrases(Côté &Robyt,1982;Figures&Edwards,1981;Zahley&Smith,1995). Thus,thestructureofeachdextranisacharacteristicofthespecific dextransucrase(Jeanesetal.,1954).Thepresentstudydiscussesthe structureofawater-solubledextranproducedbyadextransucrase elaboratedbyL.mesenteroidesFT045B.FourierTransformInfrared (FTIR) spectroscopy,1_{H Nuclear Magnetic Resonance(}1_HNMR)

spectroscopyandTLC(ThinLayerChromatography)of oligosaccha-ridesobtainedfrompolymerhydrolysisbyendodextranaseisolated fromPenicilliumspwereusedforthedeterminationofthe struc-ture.

Thenumberaveragemolecularweight(MWn)and degreeof polymerization(DPn)weredeterminedbythemeasurementofthe reducingvalueusingthecopperbicinchoninatemethodandthe measurementoftotalcarbohydrateusingthephenol–sulfuricacid method.

2. Experimental

2.1. Organism

L.mesenteroidesFT045Bwasisolatedfromanalcoholandsugar millplantandwasprovidedbytheMicrobiologyIndustrialProcess ControlDivision ofFermentec(Brazil).Thenucleotidesequence wassubmittedtotheGenBanksequencedatabase(GenBankID: JF812153.1).

2.2. Enzymes

(a)L. mesenteroides B-512FMC dextransucrase (heretofore B-512FMC dextransucrase) was obtained from a constitutive mutant of L. mesenteroides B-512FMC (Kim & Robyt, 1994; Zahley&Smith,1995)whichwasprovidedbytheLaboratory ofCarbohydrateChemistryandEnzymology,IowaState Univer-sity(USA).Theculturesupernatantwasconcentratedbypassing itthroughapolysulfoneultrafiltrationhollowfibercartridge (0.1m×1.0m,H5P100-43,Amicon,Inc.,Beverly,MA,USA)ata

flowrateof4Lmin−1_at21◦_{C.Whenthevolumeofthe}

concen-tratewasapproximately1L,smallmoleculeswereremovedby passing,(10times)through500mLof20mMpyridine/acetic acidbuffer,pH5.2,containing0.04%PVA10Kand1mMCaCl2.

Theconcentratedenzymewasthencollectedandthehollow fibercartridgewaswashedwithapproximately400mLofthe bufferwhichwasaddedtotheconcentrate(Kitaoka&Robyt, 1998).B-512FMCdextransucrase activitywas89IUmL−1_,as

determined by the 14_{C-sucrose assay (Vettori, Mukerjea, &} Robyt,2011),inwhich1.0IU=1.0␮molof␣-d_-glucose incor-poratedintodextranpermin.

(b) L.mesenteroides FT045B dextransucrase (hereinafter FT045B dextransucrase)wasobtainedfromL.mesenteroides FT045B. The growth medium for L. mesenteroides FT045 B was sucrose(40gL−1_{),yeastextract(20gL}−1_{),K2HPO4 (20gL}−1_),

CaCl2 (0.02gL−1), MgSO4 (0.2gL−1),NaCl (0.01gL−1), FeSO4

(0.01gL−1_)andMnSO

4 (0.01gL−1).Thestrainwasstoredat −20◦Cinacryogenicsolutionuntilpreparationofthe

inocu-lumfor fermentation.Thefermentationinoculum accounted for up to5% (v/v) of the total volume of the medium. The microorganismwastransferredfromthestockculturetothe samemediumandwasgrownfor18hat25◦_Cand150rpm.

The culturesupernatant wasobtained by centrifugation for 30min at 13,000×g and stored at 4◦C during the

execu-tionoftheexperiments.FT045B dextransucraseactivitywas 2IUmL−1_{,asdeterminedbythe}14_{C-sucroseassaydescribed}

above.

(c)Dextranase from Penicillium sp was obtained from Sigma [lyophilized powder, 10–25unitsmg−1 _{of solid]; one unit}

releases1.0␮moleofisomaltose(measuredasmaltose)permin

atpH6.0and37◦_{Cusingdextranassubstrate.Thepowderwas}

dissolvedinabuffersolution(about4mLof20mMPyr/Ac,pH 5.5for1mgofpowder)toobtain5UmL−1_.

2.3. Dextranreference

TheB-512FdextranwaspurchasedfromSigmaChemicalCo.

2.4. Dextranproduction

Theenzymedigestsolutionwaspreparedwiththeadditionof 40mLofbuffersolution(20mMPyr/Ac,pH5.2)to50mLof400mM sucroseandpreheatedto30◦_{Cfor10min.Theenzymereactionwas}

startedbyadding10.0mLofthedextransucrase(2UmL−1₎

pro-ducedbyL.mesenteroidesFT045Bandincubatedat30◦_Cfor33h

(2conversionperiods).Attheendofthereaction200mLofcold

ethanolwasaddedtoprecipitatethesynthesizeddextranandthe solutionwaskeptat4◦_{Cfor24h.Theprecipitateddextranwas}

cen-trifugedat13,000×gfor30minanddissolvedin100mLofwater.

Aftercomplete solubilization,thesolutionwascooledin icefor 10min.Thedextranwasprecipitatedagainwiththeadditionof 200mLofethanol,whichwasmixedandcentrifugedfor30min at13,000×g.Solubilizationandprecipitationwererepeatedonce

moretoensurethattherewasnoremainingfructoseorsucrose. Thedextranwastreatedfivetoseventimeswithacetone(30mL) andoncewithanhydrousethanol(30mL)andthenplacedintoa vacuumovenat40◦_Cfor18h.

2.5. Thenumber-averagemolecularweight(MWn)anddegreeof polymerization(DPn)

TheMWnandDPn(Jane&Robyt,1984)ofthedextranwere determined by the measurement of the reducing value using thecopperbicinchoninatemethodandthemeasurementoftotal carbohydratesusingthephenol–sulfuricacidmethod(Fox&Robyt, 1991);DPn=[(totalcarbohydratesin␮gof d_{-glucose)/(reducing} valuein␮gofmaltose)]×1.9;MWn=[(DPn)×162]+18.

2.6. Dextranhydrolysisbyendodextranase

Thedextran(10mg)wasdissolvedin960␮Lofbuffersolution

(20mMPyr/Ac,pH5.5);analiquotof40␮LofPenicilliumsp

endo-dextranase(5UmL−1_{)wasaddedandincubatedat37}◦_Cfor14h.

Aliquotsof100␮Lweretakenat10,20,30,40,50,60and840min.

Five␮Lofconcentratedtrifluoroaceticacid(TFA)wereaddedand

thealiquots(finalconcentrationof6.14MTFA)wereheatedto50◦_C

for10mintostopthereaction.

2.7. Thinlayerchromatography

Analiquotof1␮Lofeachsampleandoftheisomaltodextrin

andmaltodextrinstandardswasapplied15mmfromthebottom ofathinlayerchromatography(TLC)plate(Whatman5K)using a 5␮L Hamiltonmicrosyringe pipette. Eachspot sizewas kept

between2and3mmdistantfromeachotherbyadding1␮Lat

atime,withintervalsfordrying.Theplatewasirrigatedwiththree ascentstothetopat20–22◦_{C using,85:20:50:70(v/v/v/v)}

ace-tonitrile/ethylacetate/1-propanol/water. Afterdevelopment,the carbohydrateswerevisualizedby dippingtheTLCplateintoan ethanolsolutioncontaining0.5% (w/v)␣-naphthol and5%(v/v)

H2SO4.Afterairdrying,theTLCplatewasplacedintoanovenat

120◦_{Cfor10min.Thedensitiesofthecarbohydratespotsonthe}

TLCplateweredeterminedusinganimagingdensitometer (Bio-Raddensitometer,ModelGS-670)(Robyt&Mukerjea,1994;Robyt, 2000).

2.8. 1_{HNMRspectroscopy}

OnemilligramofdextranfromB-512Fand1mgofdextranfrom FT045Bweredissolvedseparatelyin0.5mLofpureD2Oandthen

placedinto5mmNMRtubes.1_{HNMRspectrawereobtainedfroma}

BrukerDRX500spectrometer,operatingat500MHzat25◦_C.

Ace-tone(2.22ppm)wasusedasthestandardforadjustingthe1_HNMR

chemicalshifts.

2.9. Fourier-TransformInfraredspectrometricanalysis

Fig.1. TLCanalysisofhydrolysisofdextranFT045Bwithendodextranase(5U/mL) at37◦_{C.Lane1,2,3,4,5,6,and7refertosamplestakenat10,20,30,60,90,} 120and840min,respectively;IM=isomaltodextrinstandards(IM=isomaltose, IM3=isomaltotriose,and soforth);MS=maltodextrin standards(G2=maltose; G3=maltotriose,andsoforth).

3. Resultsanddiscussion

ThedextranproducedbyL.mesenteroidesFT045Bhadanumber average molecular weight of 91,536Da and DPn of 565. Dex-tranhydrolysisbyendodextranaserequiresaminimumofsixor sevennon-substitutedcontiguouslylinked␣-(1,6)glucoseresidues

(Boune,Huston,&Weigel,1962;Côté&Robyt,1984;Richards& Streamer,1978).Thus,thedecisionwasmadetomakecertainthat L.mesenteroidesFT045Bwasproducingarealdextranwith predom-inantly␣-(1,6)-glucopyranosidiclinkageswithinthemainchains

priortocarryingouttheothercharacterizationmethods(Buchholz &Monsan,2001;Dolsetal.,1998;Seymour&Knapp,1980).

ThemixtureofoligosaccharidesobtainedfromFT045Bdextran hydrolysiswereappliedtoTLC(Fig.1)anddensitieswere deter-minedtofindthequantitativeamountofeachproductbyscanning imagingdensitometry,asdescribedbyRobytandMukerjea(1994). Thelimitofhydrolysisbyendodextranasewasexpressedasthe apparent conversioninto isomaltose.The major productsafter 840minofhydrolysiswithPenicilliumspdextranase(Fig.1,lane 7)wereisomaltose(33.2%)andisomaltotriose(24.6%).Thesewere derived fromthelinear regionsof the␣-(1,6)glucanbackbone

chains.Moreover,thebranchedoligosaccharides,representing sig-nificantbranches in dextran, branched isomaltotetraose (0.4%), isomaltopentaose (3.9%) and higher branched isomaltodextrins

Fig.2. FTIRspectraforcommercialdextranfromB512FfromFT045B.

wereobtained,indicatingFT045Bdextranbranching.Thetypeof thebranchingwasthenconfirmedbyNMRas␣-(1,3)linkage.

The type of linkages and thefunctional groups of the com-mercialdextranfromL.mesenteroidesNRRLB-512Fanddextran fromFT045BwerecharacterizedbyFTIRspectroscopicanalysis. Fig.2showstheFTIRspectraofcommercialdextranfromL. mesen-teroidesB-512FanddextranfromFT045B.Bothdextransgavevery similarspectra,indicatingasimilarstructure,althoughwith differ-entdegreesof␣-(1,3)branchlinkages.DextranfromFT045Band

B-512Fcontain␣-(1,6)linkages,asdemonstratedbythepeakat

1010cm−1_{,whichcharacterizestheconsiderablechainflexibility}

presentindextranaroundthistypeofglycosidicbond(Shingel, 2002).The ␣-glycosidicbond is also confirmed by thepeak at

916cm−1_{andthebandat1159cm}−1_{causedbycovalentvibrations}

oftheC O Cbondandglycosidicbridge.Thepeakat1111cm−1

wasduetothevibrationoftheC ObondattheC-4positionofthe glucoseresidue(Shingel,2002).Thehydroxylstretchingvibration ofthepolysaccharidecausedthebandintheregionof3410cm−1

andtheC Hstretchingvibrationcausedthebandintheregionof 2926cm−1 _{(Capeketal.,2011;Liu,Lin,Ye,Xing,&Xi,2007).The}

bandintheregionof1639cm−1 _{wasduetoboundwater(Park,} 1971).The␣-(1,6)linkages werefurtherconfirmedby1HNMR

analysis.

The1_{HNMRspectraaffordcompellingevidenceforthemain}

structuralfeaturesofdextran.Theindividualassignmentsderived fromthepresentexperimentsandthosereportedintheliterature aredisplayedinTable1.

Fig.3displaysthe1_{HNMRspectraofthenativedextranfromL.} mesenteroidesB-512FandFT045BinD2O.Theresultsshowthat

the dextran from L. m. B-512F and L.m. FT045B have a simi-lar structure, although the␣-(1,3) branch linkages and ␣-(1,6)

Table1

Assignmentsofindividualsignalsin1_{HNMRspectraofthenativedextranfrom} B-512F(usedasstandard)andfromFT045Bdextran.

B-512F(reported values)

B-512F (experimental)

FT045B (experimental)

1_H(ppm)a 1_H(ppm) 1_H(ppm)

C1 H 4.99 4.91 4.90

C2 H 3.6 3.50 3.51

C3 H 3.76 3.65 3.63

C4 H 3.52 4.45 3.42

C5 H 3.88–4.04 3.85 3.85

C6 H 3.88–4.04 3.93 3.95

C6′ _{H 3.81–3.86 3.70 3.70}

Table2

Differentlinkagestructuresfoundinalldextranseriesandreportedinliterature.

Percentofglycosidiclinkagesa

␣-1,6 ␣-1,3 ␣-1,3branch ␣-1,4branch ␣-1,2branch

Leuconostocmesenteroides

FT045B Sb _97.9e _2.1e

B-512F S 95 5

B-742 Lc _{87 13}

B-742 S 50 50

B-1355 L 95 5

B-1355 S 54 35 11

B-1299 L 66 7 27

B-1299 S 65 35

B-1498 S 50 50

B-1501 S 50 50

Streptococcusdownei

Mfe28 12 88

Mfe28 90 10

Streptococcusmutans

GS5 13 87

GS5 15 85

GS5 70 30

6715 S 64 36

6715 Id _{4 94 2}

a_{AdaptedfromMonsanetal.(2001).} b _S=soluble.

c _{L=lowsoluble.}

d _I=insoluble.

e_{Determinedinthisstudy.}

Fig.3.1_{HNMRspectraofnativedextranfromLeuconostocmesenteroidesB512-F} andFT045BinD2O.

linkagesdifferintermsofpercentage.Dextransofdifferent struc-turescanbeproducedbyspecificdextransucrases,eachexcreted byacertainbacterialstrain(Zahley&Smith,1995).Table2 dis-playsthedifferentlinkagestructuresfoundinthedextranseries producedbyLeuconostocmesenteroides,Streptococcusdownei,and StreptococcusmutansaswellasthecomparisonbetweenFT045B dextran,with97.9%␣-1,6linkagesand2.1%␣-1,3branchlinkages,

andB-512Fdextran, whichhasaboutmorethantwiceasmany

␣-1,3branchlinkages(5%).

The three signals that appear centered at ∼5.25ppm (not

shown)representimportantstructuralcharacteristicsof␣

-(1,3)-linked-d-_{glucosyl residues. The peak at 4.90ppm is due tothe} H-1ofthe␣-(1,6)glucosylresiduesofthemainchain(Purama,

Goswami,Khan,&Goyal,2009;Seymour,Knapp,&Bishop,1979). Theintegrationofsignals5.25and∼4.90givesthepercentageof

␣-(1,3)-linkedd-glucosyland _␣-(1,6)-linkedd-glucosyllinkages, respectively(Table2).

The FTIR and 1_{H NMR spectral analysesconfirmed that the}

polysaccharideproduced fromL.mesenteroides FT045Bis an␣

-(1,6) low-branched dextran, with2.1% ␣-(1,3)branch linkages,

givinganaveragechainlengthof(1/2.1)×100=48glucoseunits

betweenbranchlinkages,with97.9%␣-(1,6)linkages.The

com-mercialB-512FdextranfromSigmaexhibits5.0%branching,giving (1/5)×100=20glucoseunitsbetweenbranchlinkages,whichisa

significantdifference.Thereare21branchlinkagesforevery1000 glucoseunitsofFT045Bdextranand50branchlinkagesforevery 1000glucoseunitsofB-512Fdextran,indicatingasignificant dif-ferenceinstructure.B-512Fdextranhas5%␣-(1,3)branchlinkages

oneofthese,inwhichthebranchingislessthanhalfthatofthe B-512Fcommercialdextranoranyotherdextranthat hasbeen characterizedsofar.Thisproducesachainlengththatismorethan twicethelength(48vs.20glucoseunits,forFT045Bdextranand B-512Fdextran,respectively).Thisincreasedchainlengthshould givedifferentchemicalandphysicalproperties,incomparisonto otherdextrans.

Thediscoveryof newdextransthathave differentstructures and,hence,potentiallydifferentproperties,resultingfromdifferent structures,is important withregardto potentialnovel applica-tionsandthepotentialimprovementinoralterationstopreviously establishedapplications.

4. Conclusions

Thepresentexperimentsdemonstratedthatthedextran pro-ducedbyL.mesenteroidesFT045Bhasastructuresimilartothat ofL.mesenteroidesB-512Fdextran,butwith97.9%␣-1,6linkages

and 2.1% ␣-1,3 branch linkages, as compared to B-512F

dex-tran,whichhasmorethantwiceasmany␣-1,3branchlinkages

(5%).TheFT045Bdextranhasanaveragebranchchainlengthof 48 d-glucopyranose residues/molecule, as compared to B-512F dextran,whichthathasanaveragebranchchainlengthof20d -glucopyranoseresidues/molecule.

ThestructureoftheFT045Bdextran, with2.1%branching,is obviouslynotlinearandwouldhave12branchlinkagesper565 d-glucoseresiduesinadextranwithaMWnof91,536Da,as deter-minedinthepresentstudy.

Acknowledgements

TheauthorsaregratefultoProf.JohnF.RobytforallowingMary HelenP.B.Vettoritohaveanexternalresearchexperienceinthe LaboratoryofCarbohydrateChemistryandEnzymology, Depart-mentofBiochemistry,Biophysics,andMolecularBiology,atIowa StateUniversity,USA.

Financial support: The authors thank the FAPESP-São Paulo Research Foundation for financial support (grant number 2008/07410-1).

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