Successful preparation and characterization of biotechnological grade agarose from indigenous Gelidium amansii of Taiwan

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ContentslistsavailableatSciVerseScienceDirect

Process

Biochemistry

j our na l h o me p ag e : w w w. e l s e v i e r . c o m / l o c a t e / p r o c b i o

Short

communication

Successful

preparation

and

characterization

of

biotechnological

grade

agarose

from

indigenous

Gelidium

amansii

of

Taiwan

Tzu-Pin

Wang

a,∗

_,

_Li-Lin

_Chang

a

_,

_Sheng-Nan

_Chang

b

_,

_Eng-Chi

_Wang

a

_,

_Long-Chih

_Hwang

a

_,

_Yen-Hsu

_Chen

c,d

, Yun-Ming

Wang

e

a_Department_of_Medicinal_and_Applied_Chemistry,_Kaohsiung_Medical_University,_Kaohsiung_80708,_Taiwan b_{Cardiovascular}_Center,_National_Taiwan_University_Hospital_Yun-Lin_Branch,_Dou-Liu_640,_Taiwan c_Graduate_Institute_of_Medicine,_Kaohsiung_Medical_University,_Kaohsiung_80708,_Taiwan d_Department_of_Internal_Medicine,_Kaohsiung_Medical_University_Hospital,_Kaohsiung_80708,_Taiwan e_Department_of_Biological_Science_and_Technology,_National_Chiao_Tung_University,_Hsin-Chu_300,_Taiwan

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5September2011 Receivedinrevisedform 20December2011 Accepted21December2011 Availableonline2January2012 Keywords:

Gelidiumamansii

Biotechnologicalgradeagarose EDTA

Strong-anionexchangeresin Isopropanolprecipitation

a

b

s

t

r

a

c

t

ThispaperreportsthefirstsuccessfulpreparationofbiotechnologicalgradeagarosefromGelidium aman-siifoundinTaiwan.Thescale-efficiencypreparationwasachievedbyshorteningEDTAtreatmenttime throughdispersingG.amansiiagarinwaterinthepresenceofheatandEDTA,removingagaropectin impuritywithaheat-compatibleandstrong-anionexchangeresin,andprecipitatingagarosewitha cost-effectiveisopropanolmethod.TheyieldofagarosefrompreparedG.amansiiagarwas11.3%.The acquiredagarosehasagelstrengthof853gcm−2,asulfatecontentof0.14%,apyruvatecontentof1.03%, adegreeofelectroendosmosisof0.16andverylimitedbindingaffinitytoDNA.Theexcellentproperties ofagarosefromG.amansiiofTaiwanconfirmitspotentialdiversebiotechnologicalapplications.This innovativeagarosepreparationmethodwiththesignificantlyimprovedscale-efficiencycanbemodified forlarge-scalepreparationofagaroseforuseinbiotechnologicalindustryandbiochemicalresearch.

1. Introduction

Agaranditssub-fraction,agarose,aretwoofthemost com-monlyusedpolysaccharidemixtures obtainedfrom marinered algae(Rhodophyta),whichincludesthecommerciallyimportant generaGelidium and Gracilaria.Agarosepreparationsfrom both generahaveimportantbiotechnologicalapplications[1,2]andare widelyexploitedinthefoodindustry[2,3].GelidiumandGracilaria haveplayed akey roleintheﬁeld ofbiotechnologysinceKoch demonstrated agar was a suitable solid medium for growing microorganisms[1].For example,agarosegelelectrophoresis is currentlythepredominantmethodforroutinenucleicacid anal-ysis[1,2,4],andhasimplicationsinstudyingnucleicacid–protein interactions[5,6],proteinchemistry[7],andviralstructure[8].In addition,agarose-basedchromatographyisthepreferredmethod inbiomoleculeseparation[1,2].Moreover,agarsandagarosehave importantapplicationsinavarietyofareasincluding pharmaceu-tics,cosmetics,tissueengineering,cell-sizedliposomepreparation andcellencapsulation[2,9,10].

∗ Correspondingauthor.Tel.:+886073121101x2756;fax:+886073125339. E-mailaddress:tzupinw@cc.kmu.edu.tw(T.-P.Wang).

Theadvantagesofusingagaroseinscienceandindustryderive from its ability to form macroporous matrices with thermore-versiblehysteresisandahighgelstrength,itsnon-toxicityafter hydrogelformation,anditsmaintenanceofaminimalgel back-groundafterrapidstaininganddestaining[2,4,11].Theseessential agarosepropertiesareattributedtothealternating1,3-linked ␤-d-galactose and 1,4-linked 3,6-anhydro-␣-l-galactose repeating unitscalledagarobiose(Fig.1;R1=R2=R3=H)whichpolymerize

into a long chain [12,13]. The monosaccharideunits of natural agaroseareoftenmodifiedwithchargedgroupssuchassulfate andpyruvatewhichtransformsagaroseintoagaropectin(Fig.1). However,presenceofagaropectinisundesirablefortheagarose preparedfor biotechnologicalapplications [4,14–16].The nega-tivepropertiesofagaropectinaretheconsequenceofunsaturated chemicalbondsinthesulfate andpyruvatesubstitutions;these bondsbestowhighUVabsorptioninagarosegelsandinterferewith thedetectionofnucleicacidsafterelectrophoresis[4,15]. Addition-ally,thesesubstitutionscausesignificantbiomoleculeadsorption onagarosematrices[4,15].Agaroseusedinfundamentalresearch andbiotechnologyisrequiredtohaveaminimalamountofsulfate andpyruvatemodifications.

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Fig.1.Thegenericstructureofagaroseandagaropectincomposedofrepeating unitsofthedisaccharide,agarobiose.Thestructuraldifferencesbetweenagarose andagaropectinareindicatedbysubstitutionswithvariousfunctionalgroupsatR1,

R2andR3.

Various methods have been developed to prepare high-grade agarosefromeitherhighqualityagarsorlow-gradeagarose. Prepa-rationhasincludedfractionalprecipitationmethods[14,15,17–19] suchasisopropanol(IPA)precipitation,adsorptionmethods[20,21] suchasaluminumhydroxidetreatment,orchromatography meth-ods[14] suchas DEAEion exchangechromatography. Prepared agaroseisgenerallyevaluatedusingparameterswhichincludegel strength,thetemperaturesatwhichitsolidiﬁesandmelts,sulfate content,pyruvatecontent,andelectrophoreticpropertiesofthegel [1,15].Typically,biotechnologicalgradeagarosehasagelstrength ofatleast1000gcm−2,agelationtemperatureof36◦C,amelting temperatureof85◦C,asulfatecontentof0–0.15%(w/w),a pyru-vatecontentof0–0.1%(w/w)andadegreeofelectroendosmosis (EEO)at1.0%(w/w)agaroseconcentrationof0.04orless[15].

Asapotentialsourceforhighqualityagarose,Gelidiumamansiiis veryabundantinnortheasternTaiwanandhaslongbeenastaple intraditionalTaiwanesediets.G.amansiiagariseasilyprepared fromthe alga by heating, ﬁlteringand coolingwithout chemi-caltreatments[4],whichisinsharpcontrasttothatofGracilaria agarpreparationrequiredmorecomplicatedprocedures[22]. Var-iousG.amansiiapplicationsunderscoretheimportanceofthealgal extractsfortheTaiwanesefoodindustryandsuggestaplausible roleinthepharmaceuticalindustry[23].However,todate,there hasbeennoattempttouseTaiwaneseG.amansiitopreparehigh qualityagaroseforbiotechnologicaluse.

Inthisstudy,highqualityagarosewaspreparedfromindigenous Taiwanesealgae.Theuniqueandscale-efficientmethodforagarose preparation adoptsthree critical procedures:heatdispersionof G.amansiiforefficientEDTAchelationwhilesignificantly reduc-ingtreatmenttimefrom4dto1d,effectiveseparationofagarose fromimpuritiesbyaheat-compatibleanionexchangeresin,anda cost-effectiveIPAprecipitation.ItslowabsorptionunderUV illumi-nation,goodelectrophoreticproperties,andlackofDNAadsorption intheagarosegelareindicativeoftheexcellentchemicaland phys-icalpropertiesofagarosepreparedusingindigenousTaiwaneseG. amansii.Oursuccessfulmethodtopreparebiotechnologicalgrade agarosefromG.amansiinativetoTaiwanwiththescale-efficiency revealssignificantpotentialinbiotechnologyapplications.Finally, theinnovativepreparationmethodcanbemodifiedto accommo-dateindustrial-scaleproduction.

2. Materialsandmethods

The materials and detailed experimental methods are described in Supplementaldata.

3. Results

3.1. TemperatureandconcentrationoptimizationsofEDTA treatmentsforagarosepreparation

Twoessentialdeterminantsofagarosesuitabilityfor biotechno-logicalapplicationsaretheconcentrationofsulfateandpyruvate [15].Kirkpatricketal.basedontheiranalysismethodstoconclude thatagarosewithasulfatecontentof0–0.15%(w/w)andapyruvate contentof0–0.1%(w/w)isrequiredtoqualifyasbiotechnological grade[15].Treatingagarorlow-gradeagarosewithEDTA effec-tivelyreducestheconcentrationofbothofsulfateandpyruvate inthesampleswhenusedincombinationwithsubsequent separa-tionmethodssuchasadsorption[21]andIPAprecipitation[15].The originalmethodofBartelingused20mMEDTAintheﬁrstagarwash for2dand10mMEDTAinthesecondagarwashforanother2dat roomtemperature[21].Wereasonedthathigherconcentrationsof EDTAinthepresenceofheatwouldresultinmoreeffectiveEDTA chelationofdivalentcationsfromtheagaropectinandfacilitate preparationofbiotechnologicalgradeagarose.

Increasing both the temperature of the agar solution and theEDTAconcentrationto30mMduringincubationsignificantly reducedthecontentofsulfateandpyruvateinthepreparedagarose (Fig.2).First,45◦C wasselected astheincubationtemperature oftheagarsolutionduringEDTAtreatmentduetoa concernof energyconsumptionbytheprolongedincubation.Inaddition,the EDTAconcentrationswerechangedfrom20mMto40mMforthe firstwashand10to20mMforthesecondwash.Themethodof Bartelingrequires4dtocompletetwoEDTAwashes[21],a pro-cesstootime-consumingforindustrialprocessingrequirements. Therefore,thetimeofeachEDTAwashwasshortenedtoonly12h toallowtheoverallEDTAtreatmentstepscompletedin1d.Results indicatedthattheoptimalconditionsforagarosepreparationwere 30mMEDTAforthefirstwash,15mMEDTAforthesecondwash, and an incubationtime of 12hfor each washat 45◦C (Fig. 2). Since45◦Cmightnot betheoptimaltemperaturefor theEDTA treatment,the30mM/15mMEDTAwashstepswereconductedat severaldifferentincubationtemperaturestodeterminethe opti-maltemperaturetofurtherreducesulfateandpyruvatecontents. Incubationoftheagarsolutionat50◦Cduringthe30mM/15mM EDTAtreatmentstepprovidedthelowestagarosesulfateand pyru-vatecontent(Fig.2).Itisconcludedthatthe30mM/15mMEDTA treatmentwithanincubationtimeof12hforeachwashat50◦Cis theoptimalEDTAtreatmentmethodtoprepareG.amansiiagarose. 3.2. Biotechnologicalgradeagarosepreparationwithexcellent electrophoreticproperties

WefurthersubstitutedtheAl(OH)3adsorptionmethodwithion

exchangecolumnchromatographytoobtainagarosewithlower sulfateandpyruvatecontenttomeetthedemandsof biotechno-logicalapplications.Aprerequisitetoeffectivelyremovingmore oftheextensivelymodiﬁedagaropectinfromtheagarose prepara-tionduringionexchangecolumnchromatographyistocompletely dissolveagarose,generallyrequiredtomaintainthesolution tem-peratureover80◦C,inanaqueoussolutioncontainingionexchange resin.Thestudywasthusmotivatedtoexploitcommercially avail-able,heat-stable,andstronganionexchangeresins.Theproperties ofthehydrophilicSuperQ-650Manion exchangeresinare ideal for preparing agarose suitable for biotechnological applications (Table1).However,thesulfateandpyruvatecontentinthe pre-paredagarosewerestillnotlowenoughtomeetbiotechnological graderequirements[0.99%sulfatecontent(w/w);1.40%pyruvate content(w/w)].

Thepreparationofbiotechnologicalgradeagarosewasachieved byfurtherpurifyingagaroseusinganimprovedIPAprecipitation

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17.50% 10.00% 12.50% 15.00% 2.50% 5.00% 7.50% Contents 0.00% Original G. ammsii G. ammsii agar RT, 2d, 0.02 M EDTA only RT, 2d, 0.02 M EDTA, Al(OH)3, IPA 45°C, 12h, 0.02 M EDTA, Al(OH)3, IPA 45°C, 12h, 0.03 M EDTA, Al(OH)3, IPA 45°C, 12h, 0.04 M EDTA, Al(OH)3, IPA 50°C, 12h, 0.03 M EDTA, Al(OH)3, IPA 40°C, 12h, 0.03 M EDTA, Al(OH)3, IPA RT, 2d, 0.03 M EDTA, Al(OH)3, IPA Method of Preparation

Fig.2.SulfateandpyruvatecontentinG.amansii,G.amansiiagarandG.amansiiagarosepreparedbyvariousmethods.Thecontentofsulfate(blackbar)andpyruvate(gray bar)insamplesweredeterminedbythemethodsdetailedinSection2.Dataareexpressedasmeans±standarddeviationsoftriplicatedetermination.

Table1

ChemicalandphysicalpropertiesofbiotechnologicalgradeagarosepreparedfromTaiwaneseG.amansiicomparedtocommerciallyavailableagarose.Dataareexpressedas means±standarddeviationsoftriplicatedetermination.

Agarosesamples Sulfatecontent

(%,w/w)

Pyruvatecontent (%,w/w)

Gelstrength

(gcm−2₎ Gelling₍◦_C) temperature Melting_temperature₍◦_C) DegreeofEEO

SuperQ-650M(fromthisstudy) 0.14±0.01 1.03±0.07 853±11 34±0 75.8±0.3 0.16±0.01

SeaKemLE(Lonza) 0.35±0.01 1.07±0.10 945±16 36±1.5a,b _>90a,b _0.22_±_0.01

BIO-41027(Bioline) 5.36±0.28 0.44±0.04 154±10 37–39a _88–90a _0.18_±_0.01

a_Data_provided_by_the_{manufacturers}₍_{http://lonza.com/group/en.html}_and_{http://bioline.gene-quantiﬁcation.info/}_). b _Measured_in_1.5%_agarose_gels.

method.Kirkpatricketal.previouslydemonstratedthatthe addi-tionofIPAtoagarosesolutions(2:1volumeratioofIPAtoagarose solution)couldselectivelyprecipitateagarose[15].Withthegoal ofdeveloping a morecost-effective IPAprecipitationprocedure by decreasing volume of IPA without sacriﬁcing the efﬁciency of agarose precipitation, the volume of IPA was systematically reducedtodeterminethesmallestvolumeofIPArequiredto effec-tivelyprecipitateagarose(Fig.S1).Theresultsindicatedthat,by usinga1.5:1volumeratioofIPAtoagarosesolution,theagarose yieldswerecomparabletothatofa2:1ratio.Thus,avolumeratio ofIPAtoagarosesolutionof1.5wasdeterminedtobetheoptimal conditionfortheIPAprecipitationinpreparingbiotechnological grade agarose. The prepared agarose had sulfate and pyruvate contentsof 0.14% and1.03%, respectively (Table1 and Fig.S1). Theselevelswerelowerorcomparabletotwocommercial biotech-nologicalgradeagaroseproducts(LonzaSeaKemLE andBioline BIO-41027).Thelowsulfateandpyruvatecontentsofthe SuperQ-650M-preparedagarosealsocontributedagoodEEOvalueof0.16, whichisbetterthantwocommonlyused,commercial biotechno-logicalgradeagaroseproducts.

Theexcellentbiotechnologicalpropertiesofthe SuperQ-650M-preparedagarosewere demonstratedwhen DNA sampleswere analyzedsimultaneouslybyelectrophoresisingelspreparedfrom eithertheSuperQ-650MortheLonzaSeaKemLEagarose(Fig.3). TheSuperQ-650M-preparedagarosegelprovidedbetterDNA res-olution even through with a slightly higher background signal whencomparedtothatofthegelpreparedusingLonzaSeaKem agarose(signal intensity,quantiﬁedbyImageQuantsoftware,of 9×105_in_SeaKem_gel_and_of_1.1_×₁₀6_in_{SuperQ-650M-prepared}

gel).Moreover,netDNAbandsignalsafterelectrophoresisinthe SuperQ-650M-preparedagarosegel hadhigher intensitycounts and,thus,betterDNAdetectionsensitivitythanthatoftheSeaKem

agarose(netsignalintensityof202bp=6.6× 106 _in_the_SeaKem

gel and 7.3×106 _in _the _{SuperQ-650M-prepared} _gel _for ₂₀₂_bp

DNA).Similarly,evidenceofbetterelectrophoreticpropertiesof theSuperQ-650M-preparedagarosegelwasprovidedwhen com-paredwithelectrophoresisresultsoftheBiolineagarosegel(results notshown).TheSuperQ-650M-preparedagarosegelalsoshowed nodetectableDNAbindingability(Fig.S2).Theoutstanding elec-trophoreticresultsobtainedwhenusinggelspreparedusingthe SuperQ-650Magaroseareconsistentwiththerelativelylow sul-fateandpyruvatecontent,andalowdegreeofEEOoftheagarose, whichfurthersupportsthepotentialoftheG.amansiiagarosefor diversebiotechnologicalapplications.

Fig.3.Comparisonofexcellentgelelectrophoresispropertiesof SuperQ-650M-preparedagarose and SeaKem LEagarose. DNA usedfor gel electrophoresis containedmostly100-bpand202-bpDNAfragmentsampliﬁedfromthepGEM-T vectorasdescribedinSection2.ADNAsample(32ngof202bpand36ngof100bp) wasloadedintoeachwellof3%gelspreparedfromtheSeaKemLE(left)orfrom theSuperQ-M650-prepared(right)agaroseinTBE,andelectrophoresedinbothgels simultaneouslyinthesamestandardelectrophoresisunit.TriplicateDNAsamples wereappliedtoeachagarosegeltoensureresultreproducibility.Thetoparrow indi-catesthelocationofthe202-bpDNAmarker;thebottomarrowrepresentsmigration ofthe100-bpDNAmarkerafterelectrophoresis.

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3.3. Physicalpropertiesofthebiotechnologicalgradedagarose The physical parameters for the SuperQ-650M-prepared agarosewerecomparedtoLonzaSeaKemLEandBiolineBIO-41027 agarose(Table1).Whilethegelstrengthwasslightlylowerthan thatofSeaKemagarose,theSuperQ-650M-preparedagarosehad moredesirable lowergelling andmelting temperatures, critical propertiestoexploitagarosein nucleicacidgelelectrophoresis applications.Theappropriatephysicalpropertiesofthe SuperQ-650M-preparedagarosemeettherequirementsforapplicationsin biotechnology.

4. Discussion

Successfulpreparationofbiotechnologicalgradeagarosefrom indigenousG.amansiiofTaiwanwithscale-generatedefﬁciency wasattainedbydecreasingEDTAtreatmenttime,incorporating anovelanionexchangeresintopurifyagarosefromimpurities, anddecreasingtherequiredvolumeofIPAtoeffectivelyprecipitate agarose.WashingG.amansiiagarinthepresenceofhigherEDTA concentrations andelevated temperaturesnotonly contributed decreasesofthedegreeofEEOandthelevelsofsulfateand pyru-vateinthepreparedagarosebutalsosigniﬁcantlyreducedtheEDTA washingtimefrom4dto1dwhicheliminates3dfrompreviously reportedEDTAmethods[15,21].

Ionexchangecolumnchromatographywasindispensableinthis studytoobtainagarosesuitableforbiotechnologicalapplication. EssentialpropertiesoftheSuperQ-650Mresinallowedforamore effective separation of agaropectinfrom solutions and resulted in agarose with lower sulfate and pyruvate content and good electrophoreticproperties(Table1andFig.3).However,special precautionmustbetakenduringtheagarose-SuperQ-650Mresin incubationtopreventdamageduringmixing.Unattendedmixing whilesuspendingtheSuperQ-650Mresinintheagarosesolution canrupturetheresin beads. Resindebriscan passthroughthe ﬁlterdisk, contaminatetheagarose,anddeleteriouslyaffectthe electrophoreticpropertiesofthepreparedagarosegel.Asstatedin MaterialsandmethodsinSupplementarydata,wecarefullystirred agarosemixturesbyhandfor30mintoensuretheanionexchange resinremainedsuspendedinsolutionwhileavoidingbreakageof theSuperQ-650Mresin.Weneverobserveddebrisofthe SuperQ-650Mresininagarosepreparationifthesamevigilantmeasures weretaken.

ThedevelopmentofamethodtoreducetherequiredIPA vol-umeforeffectiveagaroseprecipitationisalsoanimportantﬁnding ofthecurrentstudy.ByusinglessIPAduringthepreparationof agarose,industryoperationcostscanbereduced.Inaddition,the improvedIPAprecipitationmethodcanbeamore environmen-tallyfriendlyprocessifIPAisaccidentallyreleasedduringindustrial agarosepreparation.

The SuperQ-650M-prepared agarose demonstrated physical propertiescomparableorbetterthantwocommerciallyavailable agarose products (Table 1). Speciﬁcally, the SuperQ-650M-preparedagarosehaslowergellingandmeltingtemperatures,and comparable gelstrengththan thoseof SeaKemLEgel. Interest-ingly, thegel strength andthe sulfate and pyruvate contentof boththeLonzaandBiolineagarose,whichweremeasuredinthe currentstudy,differedfromthosereportedbythemanufacturers. Discrepanciesinvaluesbetweenreportedphysicalandchemical parametersandthosepresentedinthisstudymaybetheresultof differingmeasurementmethods.Nostandardprotocolsforagarose characterizationhavebeenacceptedbyacademiaorindustry. Cur-rentresults,therefore,providenobasisfordisputingtheaccuracy ofagarosespeciﬁcationsdocumentedbythemanufacturers. Nev-ertheless,theSuperQ-650M-preparedagaroseunequivocallyoffers

idealpropertiesforbiotechnologicalapplications.Researchis pro-ceedingtoimproveandreﬁnetheprocedurestoenableindustrial productionofagarosefromindigenousG.amansiiofTaiwanasa waytoachieveitsfulleconomicpotential.

Authorcontributions

T.-P.W.designed,analyzeddataandwrotemanuscript;L.-L.C. performedresearchandanalyzeddata;S.-N.C.providedexpertise, performedresearchandanalyzeddata;E.-C.W.providedexpertise; L.-C.H.providedusefulreagents;Y.-H.C.provideduseful discus-sionsandanalytictools;Y.-M.W.providedusefuldiscussionsand analyzeddata.

Conﬂictofintereststatement

Nonedeclared.

Acknowledgments

TheauthorsthankYu-ZhengSuandYi-JhenLinfortechnical assistance,andDrs.SusanFetzerandScottSeveranceforcritical readingofthemanuscript.TheauthorsalsothankDr.Jenn-Shou Tsaiof theDepartmentof FoodScienceat theNationalTaiwan OceanUniversity forpreparationof theG.amansii agarand the NationalKaohsiungMarineUniversity,Kaohsiung,Taiwanfor per-missiontousetherheometertomeasuregelstrength.Thiswork wassupportedwithagrantfromtheSmallBusinessInnovation Research (SBIR) program, Ministry of Economic Affairs, Taiwan (1Z1000103) allocated to T.-P. W. The funding agency has no involvementinstudydesign;inthecollection,analysisand inter-pretation ofdata; in thewriting of themanuscript;and in the decisiontosubmitthearticleforpublication.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.procbio.2011.12.015.

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