comparison of the glucooligosaccharide profiles produced from

BASE Biotechnol. Agron. Soc. Environ.201014(4),607-616

ComparisonoftheglucooligosaccharideprofilesproducedfrommaltosebytwodifferenttransglucosidasesfromAspergillus nigerDorothéeGoffin(1,2),BernardWathelet(1),ChristopheBlecker(2),ClaudeDeroanne(2),YvesMalmendier(3),MichelPaquot(1)(1)Univ.Liege-GemblouxAgro-BioTech.DepartmentofIndustrialBiologicalChemistry.PassagedesDéportés,2.B-5030Gembloux(Belgium).E-mail:[email protected](2)Univ.Liege-GemblouxAgro-BioTech.DepartmentofFoodTechnology.PassagedesDéportés,2.B-5030Gembloux(Belgium).(3)MeurensNatural.RuedesMartyrs,21.B-4650Herve(Belgium).

ReceivedonJuly16,2009;acceptedonDecember1,2009.

Prebioticisomaltooligosaccharide(IMO)preparationscontainα-D-glucooligosaccharidesandtheirstructureisthekeyfactorfortheirprebioticpotential.Thetransglucosylationselectivityisknowntodependontheenzymespecificityandmoreover,maltoseandα-glucooligosaccharidescanactuallyactasbothglucosyldonorandacceptorinthereaction.Thus,twocommercialenzymes,aglucosyltransferaseandanα-glucosidase,weretestedaloneandincombinationonpuremaltosetostudytheirspecificitiesandtheIMOprofileobtained.Thereactionsweremonitoredusingastep-forwardAEC-PADanalyticalmethodwhichpermittedtodetectandresolvenewunknownIMO.StructuraldeterminationofunknownIMOwasattemptedusingtheirretentiontimesandrelativeabundance.Asageneralrule,theα-glucosidasehasamoreexpressedhydrolyzingactivityleadingtoproductscontaininglessresidualdigestibleα-(1-4)linkagessuchasisomaltose,isomaltotriose,isomaltotetraose,kojibioseandnigerosewhiletheglucosyltransferaseproducesimportantamountofpanose.Finally,thecombinationofthetwoenzymesleadedtoanintermediateIMOprofile.IMOsyrupscompositionwasthusprovedtobedependantonthespecificityofthetransglucosylatingenzymesothatproductsprofilescanbedesignedusingdifferentenzymesandindifferentproportion.Keywords.Transglucosylation,glucosyltransferase,glucosidase,isomaltooligosaccharides,prebiotic,AEC-PAD.

Comparaison des profils en glucooligosaccharides produits à partir de maltose par deux transglucosidases différentes d’Aspergillus niger. Les préparations prébiotiques d’isomaltooligosaccharides (IMO) contiennent des α-D-glucooligosaccharides et la structurede ceux-ci est l’élémentmajeurdéterminant le potentiel prébiotiqued’unproduit. Ilestétabliquelasélectivitédelaréactiondetransglucosylationdépenddelaspécificitédel’enzymeetquelemaltoseetlesα-glucooligosaccharidespeuventàlafoisjouerlerôlededonneuroud’accepteurderésidusglucosylauseindelaréaction.Deuxenzymescommerciales,uneglucosyltransféraseetuneα-glucosidase,ontdoncététestéesseulesouencombinaisonsurdumaltosepurafind’étudier leursspécificitéset leprofilenIMOobtenu.Lesréactionsontétésuiviesà l’aided’uneméthodeanalytiqueamélioréeAEC-PADpermettantladétectionetlarésolutiondenouveauxIMOinconnus.Ladéterminationstructuraledecesnouvellesespècesaensuiteétéentrepriseparlebiaisdeleurtempsderétentionetleurabondancerelative.Demanièregénérale,l’α-glucosidasepossèdeuneactivitéhydrolytiqueplusprononcéeproduisantdescomposéscontenantmoinsdeliaisonsrésiduellesα-(1-4)digestiblestellesquel’isomaltose, l’isomaltotriose, l’isomaltotétraose, lekojibioseetlenigérose, alorsque laglucosyltransféraseproduitdesquantités importantesdepanose.Enfin, la combinaisondesdeuxenzymesapermisl’obtentiond’unprofilenIMOintermédiaire.Lacompositiondessiropsd’IMOs’avèredoncdépendantedelaspécificitédel’enzymetransglucosylante,cequipermetdefairevarierleprofilenIMOduproduitobtenuenjouantsurlechoixdesenzymesetleurproportion.Mots-clés.Transglucosylation,glucosyltransférase,glucosidase,isomaltooligosaccharides,prébiotiques,AEC-PAD.

608 Biotechnol. Agron. Soc. Environ. 201014(4),607-616 GoffinD.,WatheletB.,BleckerC.etal.

1. IntroduCtIon

Prebiotics are non-digestible dietary componentsthatpass through thedigestive tract to thecolonandselectively stimulate proliferation and/or activityof desired populations of bacteria indigenous to thehuman or animal colon in situ (Gibson et al., 1995;Rycroft et al.,2001;Roberfroid,2008;Wang,2009).Among these prebiotics, isomaltooligosaccharides(IMO)areforyearsthemostsuccessfulonesinAsiancountriesthankstotheirmanyfavorablepropertiesforapplication in food industry (Kohmoto et al., 1988;1991; Kanno, 1990; Kaneko et al., 1995; Gu et al.,2003;Goffinetal.,2010).

Prebiotic mixtures composition is the key factorfor their efficiency throughout the structure/functionrelationshipaswellasthepossiblepartialdigestionbyendogenous enzymes that can occur before reachingthecolon(Sanzetal.,2005).Consequently,thestartingsubstrate, enzyme type and specificity together withthe enzymatic reaction precise control are criticalpoints to produce optimized IMO preparation withhighprebioticpower.Twotypesoftransglucosylatingenzymes are available toproduce IMOstarting fromhydrolyzedstarch:glucosyltransferases(EC.2.4.1.24)and α-glucosidases (EC. 3.2.1.20) (Plou et al.,2007). Glucosyltransferases catalyze group-transferreactions ofmonosaccharides even in dilute reactionconditions.TransferaseproducingIMOarenon-Leloirenzymes that require neither co-factors nor activatedsubstrates,astheydirectlyemploythefreeenergyofoligosaccharides osidic bond cleavage (Plou et al.,2002). Glycosidases are hydrolytic enzymes, whichcancatalyzeatacertainextenteitherreversehydrolysis(thermodynamic control) or transglucosylation(kinetically-controlledprocess)(Peruginoetal.,2004),since they are retaining glucosyl hydrolase (GH)(Chiba, 1997). Some α-glucosidases indeed exhibitclear transglucosylation activity (Kato et al., 2002;Faijes et al., 2007). In both cases, the non-reducingD-glucosyl residue of the donor may be transferredtowater(hydrolysis),toD-glucosylresiduesreleasedby hydrolysis, or to the non-reducing residue ofmaltose and every α-glucooligosaccharide presentin the solution.This transfer canoccur to the 6-OH,2-OH,3-OHand/or4-OHgroupof thenonreducingglucoseunitoftheacceptordependingontheenzymespecificity. Maltose and α-glucooligosaccharidescanactually act asbothglucosyldonor andacceptorin the reaction. The starting substrate is, thus, alsodeterminant because of the enzymes differentialcapacitytousevarioussubstratesasdonororacceptorinthereaction(Pazuretal.,1978;Hamadaetal.,1980;Benson et al., 1982; McCleary et al., 1989). IMOpreparationsarethuscomposedofacomplexmixtureofglucoseoligosaccharidescharacterizedatthesame

timebytheirDPvalue(from2to~10),linkagestypes[α-(1-2, 3, 4 or 6)] and the proportion and positionof each type of linkage.Numerous articles since the1950s, evaluated the production of IMO (McClearyetal.,1989;Duanetal.,1994;Yunetal.,1994)andassociatedα-glucooligosaccharides(Yamamotoetal.,2004)byenzymeswithspecificactivitiesusingmainlypaperchromatography(Barkeretal.,1953;Nishietal.,1975; Kato et al., 2002) and paper electrophoresis(Takahashi et al., 1988), orHPLCon amino columnwith RI (Duan et al., 1995; Nakanishi et al., 2006),ELSD (Fernandez-Arrojo et al., 2007) or UV withABEE-labeled oligosaccharides (Kobayashi et al.,2003)detection.However,thefirsttwomethodsdonotpermitthefulldiscriminationofallstructuralisomersandtheirquantificationwhileaminocolumnresolutionisstillverylimited.Consequently,resultsdonotreflectthefullcomplexityofthemixturesandquantificationcan be biased. It is important to specify that in thispaper, IMO definition in the strict sense [only α-(1-6) linkages] is extended and IMO are considered asα-glucooligosaccharidespossessingatleastone“ALO”linkage (Anomalously Linked Oligosaccharides,Takaku,1988)i.e.α-(1-6),α-(1-2)orα-(1-3).Indeed,these compounds are believed to participate to theprebioticeffectandareoftenconsideredinnutritionalstudies(Kanekoetal.,1994).

Therefore,inthispaper,twocommercialenzymes,aglucosyltransferaseandanα-glucosidase,aretestedalone and in combination on pure maltose to studytheiractivitiesandtheIMOprofileobtained.Inordertomonitormore accurately IMO production, a step-forwardanalyticalmethodusingAEC-PADandabletodiscriminatestructurallyclosemoleculeisused(Goffinetal.,2009a).Indeed,thismethodenablestoseparateawiderrangeofIMOandnon-IMOby-productsthanprevious methods (Koizumi et al., 1989; Ammeraalet al., 1991; Paskach et al., 1991;Vinogradov et al.,1998;Dols-Lafargueetal.,2001;Demuthetal.,2002;Nakanishietal.,2006)andthustodetectnewtypesofIMOandobtainabetterpictureoftheproductprebioticpotential.Moreover,somestructuresareproposedfortheseunknownIMOaccordingtotheirretentiontimesandrelativeabundances.Howeverthesestructureswillneed tobeconfirmed individually inacomingpaperbystructuraldeterminationmethodssuchasNMRorLC-MS.

2. ExpErIMEntal

2.1. reagents

To prepare the standard solutions, sample solutionsandthemobilephase,18MΩpurifiedwaterproducedwithalaboratorywaterpurificationsystem(Barnstead,

Transglucosylationtoproduceisomaltooligosaccharides 609

IA, USA) was used throughout the experiments.D-Glucose,maltoseandpanosewerepurchasedfromSigma-AldrichSA(Bornem,Belgium),isomaltotriose,isomaltotetraose, isomaltopentaose, isomaltohexaoseand isomaltoheptaose from Medac GmbH (Wedel,Germany), kojibiose, nigerose, maltotriose, malto-tetraose, maltopentaose, maltohexaose and malto-heptaose from Wako chemicals GmbH (Neuss,Germany), nigerotriose from Dextra LaboratoriesLTD (Reading, UK) and 6-α-D-glucosyl-maltotriosefromMegazyme (Dublin, Ireland) andwere used asreceivedandkeptdriedinadesiccatorcabinettoavoidmoistening of high DP (degree of polymerization)sugars.Thestandardmixturesolutionwascomposedofthe18sugarsinconcentrationsrangingfrom0.075to 0.25mg.ml-1. Koji-family standard molecules[α-(1→2) linkages] were prepared as described inGoffin et al. (2009a). Guarantee grade reagent 50%sodium hydroxide concentrated solution used topreparemobilephasewaspurchasedfromJ.T.BakerBV (Deventer, The Netherlands). Sodium acetatetrihydrate for analysis was purchased from MerckKGaA (Darmstadt, Germany). Two commercialenzymesweretested:theα-glucosidase(EC.3.2.1.20.)L “Amano” from Amano Enzyme INC. (Nagoya,Japan) and the glucosyltransferase (EC. 2.4.1.24)L-500 from Genencor International INC. (Leiden,The Netherlands). For the enzyme activity assays,p-nitrophenylα-D-glucosideandTrizmasolution2%w/vwerepurchasedfromSigma-AldrichSA(Bornem,Belgium).

2.2. aEC-pad analysis of transglucosylation products

The chromatography system used was an ICS-3000system (Dionex,Sunnyvale,CA,USA).This systemconsisted of an SP gradient pump system, a DCdetector/chromatographymodule thermally regulatedwith a 25µL injection loop, an AS40 autosampler,andanED40electrochemicaldetectorequippedwithan amperometric cell. The cell comprised an about1mmdiametergoldworkingelectrode,aglassandAg/AgClcombinationreferenceelectrode(Dionex)andatitaniumcounterelectrodeconsistingofthecellbody.The chromatographic separation of the sugars wasperformed on a CarboPac PA100 analytical column(250mm× 4mmi.d.Dionex)andaCarboPacPA10guardcolumn(40mm×4mmi.d.Dionex)ataflow-rate of 1ml.min-1; both columns were packed withan identical microporous, polymeric anion-exchangematerialandwereinstalledinthethermalcompartmentat a controlled temperature of 30°C. The sampleinjectionvolumewas25µl.Thegradientelutionwasperformed with mobile phases A (100mM sodiumhydroxide solution) and B (600mM sodium acetate

trihydrate in a 100mM sodium hydroxide solution)usingthegradientprogramdescribedbyGoffinetal.(2009a).

2.3. Enzymatic activities

Transglucosidicactivitiesofenzymesaredeterminedusing the method described by Megazyme withp-nitrophenylα-D-glucosideassubstrate.Instandardconditions, transglucosidic activities aremeasured at40°Cfor10minina400µlfinalvolumeof200mMacetate buffer (pH 4.0) containing 10mg of enzymesolution(withdensitiesof1.118g.ml-1and1.17g.ml-1forL“Amano”andL-500respectively)and5mMofp-nitrophenyl α-D-glucoside.Reaction is stopped byaddition of 3ml of a 2%w/v Trizma Base solution(pH 8.5). Absorbance due to yellow nitrophenolateionsreleasedisreadat400nmwithaUltraspec4000(Pharmacia Biotech) UV/visible spectrophotometer.Thespecificactivityisexpressedinenzymaticunitpermg of enzyme preparation (EU.mg-1).AnEnzymaticUnit represents 1µM of p-nitrophenol released perminute.

2.4. transglucosylation reactions

Transglucosylation enzymatic reactions take placeat60°C in0.5l reactionmediumwith0.1Msodiumacetate buffer (4.8≤pH≤5.2), 0.5ml of enzymesolution and 100g.l-1 of maltose. For the reactionwiththetwoenzymesincombination,0.25mlofeachenzymepreparationisaddedtothebufferedmedium.Evolution of the synthesis reactions is monitoredin time by sampling aliquots every 30min (2ml).Reactionsarestoppedbyheatingaliquotsinaboilingwater bath for 5min. Before analysis, samples arediluted100timesin18MΩpurifiedwater.

3. rEsults and dIsCussIon

3.1. detection of transglucosylation products

The use of pure maltose as the transglucosylationreactionsubstrate,insteadofhydrolyzedstarchusedinindustry,leadstoasimplifiedIMOprofile(Figure 1).Moreover,theuseofastep-forwardAEC-PADmethod(Goffinetal.,2009a)permittedus tobring into lightthe presence of unknown saccharides not detectedwith previous analytical methods. Structures andabbreviations of below discussed oligosaccharidesarepresentedintable 1.Saccharidesdepictedwithanasterisk(*)arepresentintheAEC-PADchromatogrambut their identity, structure and DP are onlyspeculated while unmarked saccharides correspond


Figure 1. AEC-PAD partial chromatogram ofglucosyltransferase transglucosylation products frommaltose—Chromatogramme AEC-PAD partiel des produits de transglucosylation à l’aide d’une glucosyltransférase et à partir de maltose.

Peaknumberingispresentedintable 1 — La numérotation des pics est présentée au tableau 1;Isomaltooligosaccharides(IMO)notcorrespondingtostandardmolecules — Les isomaltooligosaccharides(IMO) ne correspondant pas à des molécules standards.

1.1001.000 900 800 700 600 500 400 300 200 100

10 12,57 15 17,5 20 22,5 27,525 30Retention times (min)

- 23

2

34

56* 7

8

10*1111’*

13*14

18*16*

15

20*

22

PAD

res

pon

se (n

C)

table 1. Structures, abbreviations, retention times and DP values of known and postulated oligosaccharides present inisomaltooligosaccharides(IMO)preparations—Structures, abréviations, temps de rétention et valeurs de DP d’oligosaccharides connus ou postulés présents dans des préparations d’isomaltooligosaccharides (IMO).

peak saccharide abbreviation structure retention dp time (min) 1 Glucose Glc D-Glcp 4.50 12 Isomaltose I2 D-Glcp-(16)-α-D-Glcp 7.40 23 Kojibiose K2 D-Glcp-(1→2)-α-D-Glcp 9.21 24 Kojitriose K3 D-Glcp-(1→2)-α-D-Glcp-(1→2)-α-D-Glcp 12.95 35 Isomaltotriose I3 D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp 13.80 36 2-O-α-isomaltosyl-D-glucose* I-K2 D-Glcp-(1→6)-α-D-Glcp-(1→2)-α-D-Glcp 15.60 37 Nigerose N2 D-Glcp-(1→3)-α-D-Glcp 17.00 28 Maltose M2 D-Glcp-(1→4)-α-D-Glcp 17.70 29 Kojitetraose K4 D-Glcp-(1→2)-α-[D-Glcp-(1→2)-α]2-D-Glcp 17.90 410 6-O-α-kojibiosyl-D-glucose* K-I2 D-Glcp-(1→2)-α-D-Glcp-(1→6)-α-D-Glcp 19.70 311 Isomaltotetraose I4 D-Glcp-(1→6)-α-[D-Glcp-(1→6)-α]2-D-Glcp 20.40 411’ 6-O-α-kojibiosyl-D-isomaltose* K-I3 D-Glcp-(1→2)-α-[D-Glcp-(1→6)-α]2-D-Glcp 21.10 412 Kojipentaose K5 D-Glcp-(1→2)-α-[D-Glcp-(1→2)-α]3-D-Glcp 21.80 513 Centose* K-M2 D-Glcp-(1→2)-α-D-Glcp-(1→4)-α-D-Glcp 22.93 314 Isomaltopentaose I5 D-Glcp-(1→6)-α-[D-Glcp-(1→6)-α]3-D-Glcp 23.27 515 Panose P D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp 23.55 316 2-O-α-isomaltosyl-D-maltose* I-K-M2 D-Glcp-(1→6)-α-D-Glcp-(1→2)-α-D-Glcp- 24.20 4 (1→4)-α-D-Glcp 17 Isomaltohexaose I6 D-Glcp-(1→6)-α-[D-Glcp-(1→6)-α]4-D-Glcp 25.29 618 6-O-α-kojibiosyl-D-maltose* K-P D-Glcp-(1→2)-α-D-Glcp-(1→6)-α-D-Glcp- 25.40 4 (1→4)-α-D-Glcp 19 Maltotriose M3 D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp 26.75 320 6-O-α-isomaltosyl-D-maltose* IP D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp- 26.98 4 (1→4)-α-D-Glcp 21 Isomaltoheptaose I7 D-Glcp-(1→6)-α-[D-Glcp-(1→6)-α]5-D-Glcp 27.08 722 Nigerotriose N3 D-Glcp-(1→3)-α-D-Glcp-(1→3)-α-D-Glcp 27.95 323 4-O-α-isomaltosyl-D-maltose I-M3 D-Glcp-(1→6)-α-[D-Glcp-(1→4)-α]2-D-Glcp 29.65 424 Maltotetraose M4 D-Glcp-(1→4)-α-[D-Glcp-(1→4)-α]2-D-Glcp 31.74 425 Maltopentaose M5 D-Glcp-(1→4)-α-[D-Glcp-(1→4)-α]3-D-Glcp 35.83 526 Maltohexaose M6 D-Glcp-(1→4)-α-[D-Glcp-(1→4)-α]4-D-Glcp 40.69 627 Maltoheptaose M7 D-Glcp-(1→4)-α-[D-Glcp-(1→4)-α]5-D-Glcp 44.88 728 4-O-α-nigerosyl-D-glucose* N-M2 D-Glcp-(1→3)-α-D-Glcp-(1→4)-α-D-Glcp n.i. 3

Unmarkedsaccharidescorrespondtoknowncompounds—Les saccharides non marqués correspondent à des composés connus;*-markedsaccharidescorrespondtounknowncompoundswithonlyspeculatednames,structuresandDP—Les saccharides marqués d’un * correspondent à des composés non connus avec des noms, structures et DP proposés. DP:degreeofpolymerization— degré de polymérisation.


to known compounds for which standards exist. Inthe chromatogram of glucosyltransferase productspresentedinfigure 1,tenpeakscorrespondtoknownsaccharidesaccordingtotheirretentiontime(RT)andthemostabundantareI2,I3,M2andPwhileK2,K3,N2, I4 and N3 are present in lower but significantquantities.Ontheotherhand,atleastsevenotherpeaksbefore27mincanbepointedoutwith13*(22.93min),18* (25.4min) and 20* (26.98min) being the mostimportant.Thesameknownandunknownsaccharidesare present in the α-glucosidase chromatogram butsometimes insignificantlydifferentproportions (datanotshown).

Regarding the enzymatic reaction, α-glucosidaseand glucosyltransferase from Aspergillus nigerboth catalyze the glucose transfer mainly throughα-(1→6) linkages or in a lesser extent and in thefollowing preferred order: α-(1→2), α-(1→3) andrarely α-(1→4) linkage (Kita et al., 1991; Duan etal.,1995;Vetereetal.,2000;Yamamotoetal.,2004;Barkeretal.,1953;Pazuretal.,1977;1978;Bensonetal.,1982;McClearyetal.,1989).Consideringthesefacts and starting from maltose (M2), the enzymescan thus produce trisaccharides directly: P, in lowerproportionsK-M2andscarcelyN-M2whilereleasingaGlcresidue.AsGlcaccumulates,disaccharidescanbe formed: I2, in lower proportionsK2 and scarcelyN2.Thus,earlyinthereaction,PandI2arethemainproducts.Laterinthereaction,theproducedIMOcanbeusedasacceptorsandpotentiallyasglucosyl-donorsbut less efficiently thanM2 (McCleary et al., 1989).DisaccharidescanthenbefurthertransglucosylatedtoproducemainlyI3,K-I2andI-K2whiletrisaccharidescanbeconvertedmainlytoI-P,K-PandI-K-M2.

3.2. Endeavour of structural identification of unknown IMo

Inthissection,unknownIMOstructuresareproposedaccording to their RT. Indeed, besides analysisconditions (Cataldi et al., 2000) and degree ofpolymerization(Lietal.,1996),relativeaciditiesofthehydroxylgroups(Lee,1990;Paskachetal.,1991)andtheir accessibility to the stationary phase functionalgroups (Hardy et al., 1988) are the main factorsgoverning AEC chromatography. Oligosaccharidesretention is thus driven by linkage type and spatialconformationwhile increasingwithDP value. Thus,thepresenceofbranching,intramolecularHbridgesortheformationofsuprastructuressuchascoilforhigherDP decrease the accessibility to the –OH (Koizumietal.,1991).

3Dstructuresofsomeoligosaccharidesresumedintable 1havebeenconstructedusingSWEETIImodelingprogramwhichusesMM3forcefieldforoptimization

(Figure 2). Glucose residues in oligosaccharides arenamed respectivelyA,B,C andD starting from thereducing end. Considering DP3 oligosaccharides 3Dstructures, relative elution position of standards canbeexplainedby several structural characteristics.K3and I3 are the less retained compounds (12.95 and13.8min) while M3 and N3 (26.29 and 27.95min)presentanaccentuateretention.K3slightretentioncanbeexplained,first,bythelossoftwo2-OHwhicharethemostacidichydroxylbesides1-OH.Secondly,themoleculeexhibitsafoldedconformationstabilizedbyhydrogenbondingsB1-O/C2-OHandA1-O/C2-OHwhichlowertheacidityofbothA1-OHandC2-OHandmakethemlessaccessible.Finally,theflexibleandacid6-OHarespreadoutsidetheformed“coil”givingnoopportunityforaneffectiveinteractingsurface.Thesamespreadingoutisobservedforthe2-OHandtheC6-OHinI3whiletwo6-OHarelostinthelinkages.Moreover, the high flexibility of α-(1-6) linkagesgeneratesahighentropyandleadstotheformationofapseudo-coilstructurewhichhinderstheinteractionswiththephase(Paskachetal.,1991).Ontheotherhand,M3andN3formationleadstothelossoftwo4-OHand3-OH,respectively,whicharelessacidic.Theyarebothverylinearmolecules,thuspresentingalargesurfaceforpotentialinteraction.InM3,the1-OHandthethree2-OHareaccessibleandalignedduetothestabilizingeffectoftheH-bondingbetween3-OHand2-Oofthenext residue.The three6-OHarealsoaccessibleandalignedontheothersideofthemolecule.InN3,2-OHand 6-OH are both accessible and are alternating onboth sides every residue.Furthermore, consideringP(23.55min) and I-M3 (29.65min), these IMO bothpossess α-(1-4) linkages and a single α-(1-6) at thenon-reducingend.Thepresenceofthislinkagereducesthe retention time compared toM3 (26.29min) andM4(31.74min),because it alters the linearityof themoleculeandmoreover,anH-bondingcanbeformedbetweenA6-OHandC2-OH.ItisworthnotingthatK4,I4,K5andI5areallelutedbeforePdemonstratingtheimportanceoftheoverallstructureonretention.

Peaks6*(15.6min)and10*(19.7min)areelutedafter K3, I3 and before I4, P,M3 and N3 and bothpresent a low area.This suggests that they are bothDP3presentingα-(1-2) andα-(1-6) linkagesas theyarepresent in significant quantity and correspondingthereforetoI-K2*orK-I2*.I-K2*possessaflexibleα-(1-6)linkagewhichmakestheC-residuerelativelymobilewhilethesamelinkageinK-I2*isstabilizedbythepresenceofaH-bondbetweenC2-OandA4-OH.Moreover,A1-OHandB2-OHaciditiesinI-K2*arereducedduetoapotentialH-bondformationbetweenthese hydroxyls. InK-I2*,A 1-OH andA 2-OH areaccessibleaswellasBandC6-OH.Peak6*and10*arethusmorelikelytocorrespondtoI-K2*andK-I2*,respectively.Peak13*(22.93min)presentarelatively


highareaandiselutedafterK4andI4andjustbeforeP andM3. It should thus correspond to aDP3witha “low retention” linkage [α-(1-2) or α-(1-6)] anda “high retention” linkage [α-(1-3) orα-(1-4)].Thepeak’s high area suggests that the linkage formedquite easily by the enzyme giving no chance toα-(1-3)andα-(1-4)atthenonreducingend.K-M2*knownascentoseisthusthebestcandidateasmaltoseis the most abundant acceptor during most of thetransglucosylationprocess.Moreover,thepresenceofanH-bondbetweenC2-OandA3-OH reduces theaccessibilitytoA1-OHandthe2-OHincomparisontoP.Peaks16* (24.2min), 18* (25.4min) and20*(26.98min)areallelutedafterPandbeforeN3andI-M3,and20*isco-elutedwithM3(datanotshown).Theyare thus likely tobeDP4withmixed linkagesand products of DP3 transglucosylation. The mostabundantDP3 and in order of importance are P, I3andspeculatedK-M2*.K-I3*issusceptible tohavea short retention time and can, thus, correspond tothesmallpeak11’while I4 retention time isknown(20.4min). Other DP4 peaks can thus correspond,

and in order of importance, to I-P*,K-P* and I-K-M2*.According to abundance, peaks 16*, 18* and20* could correspond to I-K-M2*, K-P* and I-P*,respectively.Whenlookingat3Dstructures,I-K-M2hasacompactandsimilarstructureasK-M2*withanon-reducingresiduelinkedbyaflexibleα-(1-6).ItsretentiontimeshouldthereforebeshorterthanthoseofK-PandIPinthesamewayasforK-M2*andP.When comparing I-P* and K-P*, the first exhibitsa more linear structure while the second showsrestrictedaccessibilityduetothepresenceofseveralH-bonds.

3.3. study of the reaction products of two types of transglucosylating enzymes on maltose

Twocommercialenzymes,L-500,aglucosyltransferase(EC.2.4.1.24),andL-AMANO,anα-glucosidase(EC.3.2.1.20), were tested alone and in combination onpuremaltose for their transglucosylation specificity.Their activities were, respectively, 3.79 and 4.52

Figure 2. 3DstructureofDP3standardmoleculesandsomedeterminedisomaltooligosaccharides(IMO)—Structures 3D de molécules standards de DP3 et d’isomaltooligosaccharides(IMO) déterminés.*:proposedidentification—proposition d’identification;:hydrogenbonding—ponts hydrogène;:linkagewhichpermitsarelativelyhighrotationalfreedomtotheattachedresidues—liaisons permettant une liberté rotationelle relativement importante entre les résidus correspondants.

K3 I3

P M3N3

I-P*

K-P*

K-M2

I-K-M2*

I-K2*

K-12*


EU.mg-1ofcommercialpreparation.Theevolutionofthe different products (exceptmaltose) is presentedinfigure 3. Indeed, saccharides can successivelybeproduced,increasingtheirrelativeabundanceorusedas donor (hydrolyzed) and as acceptor, decreasingtheirrelativeabundance.

OnlyIMOpresentedintable 1andcorrespondingto standards or unknown IMO described in section3.2. and inside the LOQ are considered. However,otheroligosaccharidesofDP≥4onlyrepresent2to3%ofthetotaloligosaccharides,astransglucosidasefromAspergillus nigeronlyactsonoligosaccharideswith a low degree of polymerization (Pazur etal., 1978; Benson et al., 1982; McCleary et al.,1989).Areas were corrected according to responsefactors determined from the standard moleculescalibration curves. Theoretical response factors ofother oligosaccharideswere calculated according totheir retention times and as a consequence to theirDP and linkage types (data not shown). Indeed, asretention times, response factors are linked to theoligosaccharidestructurebytheirrelativeacidityandaccessibility of their hydroxyl group andmoreover,theirdiffusioncoefficientintheelectrode.Concerningthemostabundantoligosaccharides[Figure 3(1)],notsurprisingly, the α-glucosidase (L-AMANO) has astrongerhydrolyzingactivityandworksmuchfaster.Indeed, after only 30min, it produces similar and

highamountsofPandI2,evenmoreGlcandsomeI3while the glucosyltransferase (L-500) produces lessP,GlcandlowamountsofI2andI3comparedtotheα-glucosidase.ThenL-AMANOrapidlyhydrolyzesPandproducesmoreI2andI3whileL-500continuestoproducePandincreasingamountsofI2andI3.After150min,L-AMANOonlyproducesmoreGlcwhileI2andI3contentsstayrelativelyconstantindicatingthat the enzyme easily hydrolyzes α-(1-4) linkagesof maltose but also α-(1-6) linkages presenting aneighboringα-(1-4)suchas inP.ForL-500,Ponlystarts to be hydrolyzed.At the end of the reaction,I2 and I3 contents were significantly higher forL-AMANOthanforL-500.

Similar observations can be done for minorproducts [Figure 3(2)]. For L-AMANO, smallamountsofthreeunknownIMOspeculatedasI-K2*,I-K-M2*, K-I2*, respectively and K3 and N3 areproducedwhileapproximately3%ofI4,N2andK2areproducedandnothydrolyzed.Ontheotherhand,relatively high amount of two other unknown IMOspeculatedasK-P*(4.47%)andK-M2*(3.16%)areproducedbuteasilyhydrolyzedespeciallyK-P*asforP.ForL-500,productsareagainlesseasilyhydrolyzedexcept for speculated K-I2*. Indeed, speculatedK-M2* and K-P* are still present at, respectively,2.82 and 2.70% after 310min. As a general rule,theα-glucosidasehasamoreexpressedhydrolyzing

Figure 3.Evolutionoftransglucosylationmajor(1)andminor(2)productsusingaglucosyltransferase(A)oranα-glucosidase(B)—évolution des produits majeurs (1) et mineurs (2) de transglucosylation à l’aide d’une glucosyltransférase (A) et d’une α-glucosidase (B).

A:L-500;B:L-AMANO;♦: Glc;:P;:I2;:I-P*;:I3;:K-P*;-:K12*;:K-M2*;:I4;:I-K2*;:I4;×:I-K-M2*;:K3;:N2.*Proposedidentification—identification proposée. (*) Percentage:percentageofeachspeciesinthemediumduringtheenzymaticreaction—pourcentage de chaque espèce dans le milieu au cours de la réaction enzymatique.

+x

A

B

Enzymatic reaction time (min)


Per

cent

age

( *)

Per

cent

age

( *)

Per

cent

age

( *)

Per

cent

age

( *)



(1)

(1)

(2)

(2)

50

50 50

500

0 0

00

0

0

0

3

30

15

40

2025

4

2

20

10

2

1

10

5

100

100 100

100150

150 150

150200

200 200

200250

250 250

250300

300 300

300


activity leading to products containing less residualdigestibleα-(1-4)linkagessuchasI2,I3,I4,K2andN2whiletheglucosyltransferaseproducesimportantamounts of P, I2 and speculated K-M2*, I-P* andK-P*. IMOprofilescan, thus,beorientated throughtheenzymechoice.

When using the two enzymes in combination(Figure 4), after 160min an intermediate profile isobtained, eachproduct beingpresent inpercentagesinbetweenthevaluesobtainfor theenzymesalone.Concerning the IMO content after 160min, 66.78,59.86and64.81%ofIMOarefoundforL-AMANO,L-500and their combination, respectively.Thus theenzymes do not seem to show a synergistic effectneither an inhibiting effect on eachother.However,similarmaximumIMOcontentsof67.65and66.62%areobtainedafter130and310minforL-AMANOandL-500, respectively, even though, IMOpreparationsproduced using L-AMANO contain less digestibleIMO as they present less residual α-(1-4) linkages.This confirms the fact that L-AMANO is workingmuchfasterandreactionsmustbestoppedontimetoavoid hydrolysis of produced IMO.Moreover, after160min or at themaximum IMO content (130minforL-AMANOand310minforL-500),L-AMANOpresentsahighercontentinGlcandalowercontentofM2comparedtoL-500.ThisisworthnotingbecauseGlc iseasier toeliminate fromthe IMOpreparationbyyeastfermentation(Panetal.,2005)orconversiontoprebioticgluconicacid(Goffinetal.,2009b)thanM2inordertoobtainbetterprebioticindex.

4. ConClusIon

In this paper, transglucosylation reaction leading toIMO production has been studied on pure maltoseusing two types of enzymes: a glucosyltransferase(EC.2.4.1.24) and an α-glucosidase (EC.3.2.1.20).The reactions were monitored using a step-forwardAEC-PAD analytical method which permitted todetect and resolve new unknown IMO. StructuraldeterminationofunknownIMOwasattemptedusingtheir RT and relative abundance in relation withthe enzyme propensity to form different linkages.Two different IMO profiles were found for the twoenzymesdemonstratingtheimportanceoftheenzymespecificity on the final product. As a general rule,theα-glucosidasehasamoreexpressedhydrolyzingactivity leading to products containing less residualdigestibleα-(1-4)linkagessuchasI2,I3,I4,K2andN2while theglucosyltransferaseproducesimportantamountsofPandspeculatedunknownIMOK-M2*,I-P*andK-P*respectively.Finally, thecombinationof the two enzymes leaded to an intermediate IMOprofile. Consequently, this paper permitted us, first,todetect thepresenceofunidentifiedIMOthanks totheuseofastep-forwardchromatographicmethodandthereafter proposing possible structure in relation totheirretentiontimesandabundance.Moreover,IMOsyrups composition was proved to be dependant onthe specificity of the transglucosylating enzyme sothatproductsprofilescanbethereforedesignedusingdifferentenzymesandindifferentproportion.

Figure 4. Oligosaccharidecompositionafter160minof transglucosylationwithL-500,L-AMANOor thecombinationofthetwoonmaltose—Composition en oligosaccharides après 160 min de réaction de transglucosylation à l’aide de L-500, L-AMANO ou la combinaison des deux sur le maltose.

combination

L-AMANO

L-500

Glc

30

25

20

15

10

5

Pou

rcen

tage

0I2 K2 K3 I3 I-K2* N2 M2 K-I2* I4 K-M2* P I-K-M2* K-P* I-P*


abbreviations

ABEE:p-aminobenzoicethylesterAEC-PAD: Anion exchange chromatography-PulsedamperometrydetectionALO:AnomalouslylinkedoligosaccharidesIMO:isomaltooligosaccharidesDP:degreeofpolymerizationRT:retentiontimesLOQ:limitsofquantificationEU:enzymaticunits3D:3dimensionNMR:nuclearmagneticresonanceLC-MS:liquidchromatography–massspectrometry

acknowledgements

This work was jointly supported by the Ministry of theWalloon Region and the company Meurens NaturalthroughouttheFIRSTDEIIMOBIOSEresearchproject.

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comparison of the glucooligosaccharide profiles produced from

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