FedericaDeLisea,1,FrancescaMensitieria,1,VincenzoTarallob,NicolaVentimigliaa,
RobertoVinciguerrab,AnnabellaTramicec,RobertaMarchettib,ElioPizzoa,
EugenioNotomistaa,ValeriaCafaroa,AntonioMolinarob,LeilaBirolob,
AlbertoDiDonatoa,VivianaIzzod,∗
aDipartimentodiBiologia,UniversitàFedericoIIdiNapoli,ViaCinthiaI,80126Napoli,Italy bDipartimentodiScienzeChimiche,UniversitàFedericoIIdiNapoli,viaCinthiaI,80126Napoli,Italy
cIstitutodiChimicaBiomolecolare,ConsiglioNazionaledelleRicerche,ViaCampiFlegrei34,80072Pozzuoli(NA),Italy
dDipartimentodiMedicina,ChirurgiaeOdontoiatria“ScuolaMedicaSalernitana”,UniversitàdegliStudidiSalerno,viaS.Allende,84081Baronissi(SA), Italy
a r t i c l e i n f o
Articlehistory: Received29July2016 Receivedinrevisedform 23September2016 Accepted3October2016 Availableonline3October2016 Keywords: !-l-Rhamnosidases Flavonoids Novosphingobiumsp.PP1Y Glycosylhydrolases Biotransformation a b s t r a c t
!-l-Rhamnosidases(!-RHAs)areagroupofglycosylhydrolasesofbiotechnologicalpotentialinindus- trialprocesses,whichcatalyzethehydrolysisof!-l-rhamnoseterminalresiduesfromseveralnatural compounds.Anovel!–RHAactivitywasidentifiedinthecrudeextractofNovosphingobiumsp.PP1Y, amarinebacteriumabletogrowonawiderangeofaromaticpolycycliccompounds.Inthiswork,this !-RHAactivitywasisolatedfromthenativemicroorganismandthecorrespondingorfwasidentified inthecompletelysequencedandannotatedgenomeofstrainPP1Y.Thecodinggenewasexpressedin Escherichiacoli,strainBL21(DE3),andtherecombinantprotein,rRHA-P,waspurifiedandcharacterized asaninvertingmonomericglycosidaseofca.120kDabelongingtotheGH106family.Abiochemicalchar- acterizationofthisenzymeusingpNPRassubstratewasperformed,whichshowedthatrRHA-Phada moderatetolerancetoorganicsolvents,asignificantthermalstabilityupto45◦Candacatalyticefficiency, atpH6.9,significantlyhigherthanotherbacterial!-RHAsdescribedinliterature.Moreover,rRHA-Pwas abletohydrolyzenaturalglycosylatedflavonoids(naringin,rutin,neohesperidindihydrochalcone)con- taining!-l-rhamnoseboundto"-d-glucosewitheither!-1,2or!-1,6glycosidiclinkages.Datapresented inthismanuscriptstronglysupportthepotentialuseofRHA-Pasabiocatalystfordiversebiotechnological applications.
©2016ElsevierB.V.Allrightsreserved.
1. Introduction
!-l-Rhamnosidases (!-RHAs)area group ofglycosyl hydro- lases(GHs)that catalyzethehydrolysis of terminalresidues of
Abbreviations: !-RHAs,!-l-rhamnosidases;GHs, glycosylhydrolases;GTs, glycosyl transferases; PPMM, potassium phosphate minimal medium; pNPR, p-nitrophenyl-!-l-rhamnopyranoside;MOPS,3-(N-morpholino)propanesulfonic acid;pNP,p-nitrophenolate;BSA,bovineserumalbumin;LB,LuriaBertanimedium; LB-N,LuriaBertanimediumcontainingafinalconcentrationof0.5MNaCl;LB-BS, LuriaBertanimediumsupplementedwith1mMofbothbetaineandsorbitol;LB- NBS,LuriaBertanimediumcontainingafinalconcentrationof0.5MNaCland1mM ofbothbetaineandsorbitol;IPTG,Isopropyl"-d-1-thiogalactopyranoside.
∗ Correspondingauthor.
E-mailaddress:vizzo@unisa.it(V.Izzo). 1 Theseauthorsequallycontributedtothework.
!-l-rhamnosefromalargenumberofnaturalcompounds[1].L- Rhamnoseiswidelydistributedinplantsascomponentofflavonoid glycosides,terpenylglycosides,pigments,signalingmolecules,and incellwallsasa componentofcomplex heteropolysaccharides, suchasrhamnogalacturonanandarabinogalactan-proteins[2–7]. In bacteria, l-rhamnose appears to be included in membrane rhamnolipids[8,9]andpolysaccharides[10].Accordingtothesim- ilaritiesamongtheiraminoacidicsequences,!-RHAsaregrouped intheCAZy(carbohydrate-activeenzymes)database(www.cazy. org)intofourdifferentfamilies:GH28,GH78,GH106,andNC(non- classified).
Inthelastdecade,!-RHAshaveattractedagreatdealofatten- tionduetotheirpotentialapplicationasbiocatalystsinavarietyof industrialprocessesandinparticularinthefoodindustry[1].Sev- eraldietaryproductsarerichinglycosylatedflavonoidsthatshow thepresenceofeitherarutinoside(6-!-l-rhamnosyl-"-d-glucose) http://dx.doi.org/10.1016/j.molcatb.2016.10.002
F.DeLiseetal./JournalofMolecularCatalysisB:Enzymatic134(2016)136–147 137 or aneohesperidoside (2-!-l-rhamnosyl-"-d-glucose)disaccha-
ridicunit.Inparticular,naringin,hesperidinandrutin,flavanone glycosidesfoundingrapefruitjuices,lemons,sweetorangesand vegetables,havegainedincreasingrecognitionfortheirpotential antioxidant,antitumorandanti-inflammatoryproperties[11–14]. Theabilitytohydrolyzeglycosylatedflavonoidshasbeenused tomitigatethebitternessofcitrusjuices,whichisprimarilycaused bynaringin.Rhamnoseremovalfromnaringinallowssofteningthe bittertasteof citrus juice [15,16]. Moreover,the corresponding de-rhamnosylatedcompound,prunin,isendowedwithantimicro- bialproperties[14],andshowsanimprovedintestinalassimilation whencomparedtonaringin.Otherapplicationsof!-RHAsaregain- ingpopularityintheoenologicalindustry,wheretheseenzymesare usedtohydrolyzeterpenylglycosidesandenhancearomainwine, grapejuicesandderivedbeverages[17–19].
Applicationof!-RHAstoimproveflavonoidsbioavailabilityhas alsobeenrecentlydescribed[20].Inhumans,flavonoidsabsorp- tionoccurs primarilyinthesmallintestine where theattached glucose(orpossiblyarabinoseorxylose)isremovedbyendoge- nous"-glucosidases[21–23].Terminalrhamnoseisnotasuitable substrateforhuman"-glucosidases.Therefore,unabsorbedrham- nosylatedflavonoidsarriveunmodifiedinthecolon,wherethey arehydrolyzedby!-rhamnosidaseactivitiesexpressedbythelocal microflora[24].Toimproveintestinalabsorptionofrhamnosylated flavonoids,andthustheirbioavailabilityinhumans,aremovalof theterminalrhamnosegroupcatalyzedby!-RHAswouldbeindeed beneficial[25–27].
Theabsenceofhuman!-RHAshasbeenthekeytothedevel- opmentofa noveltargeteddrugdeliverystrategy, indicatedas LEAPT(Lectin-directedenzymeactivatedprodrugtherapy)[28,29], abipartitesystembasedontheinternalizationofanengineered !-RHAbearingaglycosidicmoietythatisrecognizedbyspecific lectinspresentonthesurfaceofdifferenteukaryoticcelllines.Inthe LEAPTsystem,theintakeofarhamnosylatedprodrug,whichcan- notbeprocessedbymammalianenzymes,allowsasite-selective actionofthedrugincellswheretheengineered!-RHAhasbeen prelocalized.
Todate,microbial!-RHAshavebeenmainlypurifiedfromfun- galstrainssuchasPenicilliumand Aspergillus[30–32]; onlyone exampleof!-RHAisolatedfromaviralsourcehasbeendescribed
[33],and itis noteworthythat onlyalimited numberofbacte- rialrhamnosidaseshasbeenfullycharacterized[34–39].Oneof themaindifferencesbetweenfungalandbacterial!-RHAsistheir different optimalpHvalues, with thefungal enzymesshowing moreacidicpHoptimawhencomparedtothebacterialcounter- parts,forwhichneutralandalkalinevalueshavegenerallybeen described.Thischaracteristicsuggestsdiverseapplicationsforfun- galand bacterialenzymes,makingbacterial!-RHAssuitable in biotechnologicalprocessesrequiringgoodactivityinmorebasic solutionssuchas,forexample,theproductionofL-rhamnosefrom thehydrolysisofnaringinorhesperidin,flavonoidswhosesolubil- itystronglyincreasesathigherpHvalues[40].Inaddition,bacterial rhamnosidasescouldbeidealcandidatesfordietarysupplements havingactivity acrosstheentire gastrointestinal(GI) tract,and morespecificallyinthesmallintestinewhereflavonoidsabsorp- tionshouldmostlyoccurtoenhancethebeneficialeffectofthese moleculesonhumanhealth.
Inordertoelucidatetherealbiotechnologicalpotentialofbac- terial!-RHAs,further investigationisundoubtedlyneeded.Few detailsonthecatalyticmechanismofbacterial!-RHAsareavail- able, and most importantly, to the best of our knowledge, no attemptto improvethecatalytic efficiencyor modifysubstrate specificityoftheseenzymesbymutagenesishasbeenperformed yet.Thisisinpartconsequentialtothefactthatonlyaverylimited numberofcrystalstructuresof!-RHAsarecurrentlyavailable,such asthe!-l-rhamnosidaseB(BsRhaB)fromBacillussp.GL1[41],and
the!-l-rhamnosidasefromStreptomycesavermitilis[42].There- fore,itisevidentthatbacterial!-RHAsrepresentayetunexplored reservoirofpotentialbiocatalystsforwhichmorefunctionaland structuraldataarerequired.
AmemberoftheorderoftheSphingomonadales,recentlyiso- lated and microbiologically characterized, Novosphingobium sp. PP1Y [43,44], appears tobe a valuable sourcefor the isolation of !-RHA activities. Sphingomonadales are a group of Gram- negative!-proteobacteriawhosegenomesshowthepresenceof a great abundance of both glycosyl hydrolases (GHs) and gly- cosyltransferases(GTs).Theseactivitiesareprobablyinvolvedin the biosynthesis of complex extracellular polysaccharides and microbialbiofilms[45].TheinterestforNovosphingobiumsp.PP1Y carbohydrate-active enzymeshasgrownas thesequencingand annotationofthewholegenome,recentlycompleted,allowedthe identificationofagreatnumberofgenesencodingforbothGHs(53 orfs)andGTs(57orfs)[46].
Recently,a!-RHAactivityinNovosphingobiumsp.PP1Ycrude extractwasdescribed,whichshowedanalkalinepHoptimumand amoderatetolerancetoorganicsolvents[47].Novosphingobiumsp. PP1Ycrudeextractexpressingthisenzymaticactivitywasusedfor thebioconversionofnaringin,rutinandhesperidin.Basedonthese preliminaryresults,amoredetailedbiochemicalcharacterization ofthe!-RHAactivitywasessential.
In thiswork,theisolation, recombinantexpression andpar- tialcharacterizationofa!-RHAfromNovosphingobiumsp.PP1Yis reported.Thisenzyme,namedRHA-P,belongstotheGH106family
[48],asubgroupforwhich,accordingtoourknowledge,nocrystal structureisavailableyet.Asevidentfromouranalyses,RHA-Pisa promisingcandidateforseveralbiotechnologicalapplications.
2. Materialsandmethods 2.1. Generals
Generalmolecularbiologytechniqueswereperformedaccord- ing to Sambrook et al. [49]. Bacterial growth was followed by measuring the optical density at 600nm (OD600). pET22b(+)
expressionvectorandE.colistrainBL21(DE3)werefromAmersham Biosciences;E.colistrainTop10waspurchasedfromLifeTechnolo- gies.N.sp.PP1Ywasisolatedfrompollutedseawaterintheharbor ofPozzuoli(Naples,Italy)aspreviouslydescribed[43].
ThethermostablerecombinantDNApolymeraseusedforPCR amplificationwasTAQPolymerasefromMicrotechResearchProd- ucts. dNTPs, T4 DNA ligase, and the Wizard PCR Preps DNA purificationsystemforelutionofDNAfragmentsfromagarosegels werepurchasedfromPromega.TheQIAprepSpinMiniprepKitfor plasmidDNApurificationwasfromQIAGEN.Enzymesandother reagentsforDNAmanipulationwerefromNewEnglandBiolabs. OligonucleotidessynthesisandDNAsequencingwereperformed byMWG-Biotech.N-terminusofrRHA-PwassequencedbyPro- teomeFactoryAG.Thepresenceandlocationofapotentialsignal peptidecleavagesiteontheaminoacidicsequenceofRHA-Pwas analyzedusingSignalP4.1server(http://www.cbs.dtu.dk/services/ SignalP).
Q-Sepharose Fast Flow and p-nitrophenyl-!-l- rhamnopyranoside (pNPR)were from SigmaAldrich; Sephacryl S200HighResolutionwaspurchasedfromAmershamBiosciences. IPTG(isopropyl"-d-1-thiogalactopyranoside)wasobtainedfrom Applichem.
SolventsusedinenzymaticassayswereeitherfromApplichem (DMSO)orfromRomil(acetone).TLCsilicagelplateswerefromE. Merck(Darmstadt,Germany).
138 F.DeLiseetal./JournalofMolecularCatalysisB:Enzymatic134(2016)136–147 2.2. GrowthofNovosphingobiumsp.PP1Ycells
N.sp.PP1YcellsweregrowninPotassiumPhosphateMinimal Medium(PPMM)at30◦Cfor28hunderorbitalshakingat220rpm.
Apre-inoculuminLBwaspreparedbytransferring50#Lfroma glycerolstockstoredat− 80◦Ctoa50mLFalcontubecontaining
12.5mLofsterileLB.Thepre-inoculumwasallowedtogrowat 30◦CO/Nunderorbitalshakingandthenusedtoinoculate1Lof
PPMMataninitialcellconcentrationof0.01-0.02OD600.0.3mM
naringinewasaddedtothemediumandusedasaninducerofthe !-RHAactivity,aspreviouslydescribed[47].
2.3. CloningoforfPP1YRS05470andconstructionofthe pET22b(+)/rha-pexpressionvector
GenomicDNA wasextracted froma50mLsaturatedculture of N. sp. PP1Y as described elsewhere [44]. OrfPP1YRS05470 coding for the !-RHA activity was amplified in two contigu- ous fragments, owing to the considerable length of the orf (3441bp). The first fragment, named rha-up (1816bp), was amplifiedusinganinternaldownstreamprimer,RHA-Intdw(5′-
AGGCGGCCATGGGAATGT-3′),whichincludedaninternalNcoIsite
alreadypresentinorfPP1YRS05470,andanupstreamprimer,RHA- up(5′-GGGAATTCCATATGCCGCGCCTTTCGCT-3′),designedtoadd
aNdeIrestrictionsiteat5′ oforfPP1YRS05470.Thesecondhalf
of thegene, named rha-dw(1625bp), wasamplifiedusing the upstreamprimerRHA-Intup(5′-ACATTCCCATGGCCGCCT-3′),com-
plementarytoRHA-Intdw,andthedownstreamprimerRHA-dw (5′-AAAACCGAGCTCTCAATGCCCGCCCGTG-3′)thatwasintendedto
incorporateaSacIrestrictionsitedownstreamoftheamplifiedorf. Theamplifiedfragments,rha-upandrha-dw,werepurifiedfrom agarosegel,digested, respectively,withNdeI/NcoIandNcoI/SacI, andindividuallyclonedinpET22b(+)vectorpreviouslycutwith thesameenzymes.
Ligatedvectorswereusedtotransform E.coli,strainTop10, competent cells. The resulting recombinant plasmids, named pET22b(+)/rha-up and pET22b(+)/rha-dw, wereverified by DNA sequencing. Next, the construction of complete rha-p gene in pET22b(+) was performed. First, both pET22b(+)/rha-dw and pET22b(+)/rha-upweredigestedwithNcoI/SacIrestrictionendonu- cleases to obtain, respectively, fragmentrha-dw and linearized pET22b(+)/rha-up.
Digestionproductswerepurifiedfromagarosegelelectrophore- sis,elutedandligated.LigationproductswereusedtotransformE. coliTop10competentcellsandtheresultingrecombinantplasmid, namedpET22b(+)/rha-pwasverifiedbyDNAsequencing. 2.4. ˛-l-Rhamnosidaserecombinantexpression
ProteinexpressionwascarriedoutinE.coliBL21(DE3)strain transformedwithpET22b(+)/rha-pplasmid.
Allthemediadescribedinthisparagraphcontained100#g/mL ofampicillin.
2.4.1. Analyticalexpression
E.coli BL21(DE3) competentcells transformed withplasmid pET22b(+)/rha-pwereinoculatedina sterile50mLFalcontube containing12.5mLofeitherLB[50]orLBcontainingafinalcon- centrationof0.5MNaCl(LB-N).Cellsweregrownunderconstant shakingat37◦Cupto0.6–0.7OD600.Thispreinoculumwasdiluted
1:100in12.5mLofeitheroneofthefourfollowingmedia:LB,LB-N, LBsupplementedwith1mMofbothbetaineandsorbitol(LB-BS),or LBcontainingafinalconcentrationof0.5MNaCland1mMofboth betaineand sorbitol(LB-NBS).Cellsweregrownunderconstant shakingat37◦Cupto0.7–0.8OD600.RHA-Precombinantexpres-
sionwasinducedwith0.1mMIPTGateither23◦Cor37◦C;growth
wascontinuedin constantshaking for 3h. Cellswere collected bycentrifugation(5,524×gfor15minat4◦C)andsuspendedin
25mMMOPSpH6.9atafinalconcentrationof14OD600.Cellswere
disruptedbysonication(12timesfora1-mincycle,onice)andan aliquotofeachlysatewascentrifugedat22,100×gfor10minat 4◦C.BothsolubleandinsolublefractionswereanalyzedbySDS-
PAGE.Thesolublefractionwasassayedforthepresenceof!-RHA enzymaticactivity.
2.4.2. Largescaleexpression
Freshtransformedcellswereinoculatedinto10mLofLB-Nand incubatedinconstantshakingat37◦CO/N.Thepreinoculumwas
diluted1:100infour2LErlenmeyerflaskscontainingeach500mL ofLB-NBSandincubatedinconstantshakingat37◦Cupto0.7–0.8
OD600.
Expression of the recombinant protein, named rRHA-P, was inducedwith0.1mMIPTGandgrowthwascontinuedfor3hat 23◦C.Cellswerecollectedbycentrifugation(5,524×gfor15min
at4◦C)andstoredat− 80◦Cuntilneeded.
2.5. Nativeandrecombinant˛-l-rhamnosidasepurification Bothnativeandrecombinant RHA-Pwerepurified following threechromatographicsteps.Cellpastewassuspendedin25mM MOPSpH6.9,5%glycerol(bufferA),atafinalconcentrationof100 OD600and cellsweredisruptedbysonication(10timesfora1-
mincycle,onice).Celldebriswereremovedbycentrifugationat 22,100×gfor60minat4◦Candthesupernatantwascollectedand
filteredthrougha0.45#mPVDFMilliporemembrane.
Afterwards,cellextractwasloadedontoaQSepharoseFFcol- umn(30mL)equilibratedinbufferA.Thecolumnwaswashedwith 50mLofbufferA,afterwhichboundproteinswereelutedbyusinga 300mLlineargradientofbufferAfrom0to0.4MNaClataflowrate of15mL/h.Thechromatogramwasobtainedbyanalyzingfractions absorbanceat$=280nmandthepresenceoftherecombinant!- RHAactivitywasdetectedusingthepNPRassay.Relevantfractions wereanalyzedbySDS-PAGE,pooledandconcentratedatafinalvol- umeof∼0.5mLusinga30kDaAmiconultramembrane,Millipore. ThesamplewasthenloadedontoaSephacrylHRS200equilibrated withbufferAcontaining0.2MNaCl(bufferB).
Proteinswereelutedfromthegelfiltrationcolumnwith250mL ofbufferBataflowrateof12mL/h.Fractionswerecollected,ana- lyzedandscreenedforthepresenceof!-RHAactivityaspreviously described.Atthisstage,NaClwasremovedfrompooledfractions byrepeatedcyclesofultrafiltrationanddilutionwithbufferA.The samplewasthenloadedonaQSepharoseFFcolumn(30mL)equi- libratedinbufferA.Thecolumnwaswashedwith50mLofbuffer A,afterwhichboundproteinswereelutedwith300mLofalinear gradientofbufferAfrom0to0.25MNaClataflowrateof13mL/h. Fractionswerecollected,analyzedbySDS-PAGEandscreenedfor thepresenceof!-RHAactivity.Relevantfractionswerepooled, concentrated,purgedwithnitrogen,andstoredat− 80◦Cuntiluse.
2.6. Analyticalgelfiltration
Analyticalgel-filtrationexperiments werecarriedout asfol- lows:100#Lofa40pMproteinsamplewasloadedonaSuperdex 200HR10/300columnpreviouslyequilibratedinbufferB,installed onanAKTATMFPLCTM(GEHealthcareLifeScience).Sampleswere
elutedisocraticallyatRTataflowrateof0.5mL/min.Proteinelu- tionwasmonitoredat$=280nm.Amolecularweightcalibration wasperformedinthesamebufferwiththefollowingproteinsof knownmolecularweight:"-amylase(200kDa),glyceraldehyde-3- phosphatedehydrogenase(143kDa),carbonicanhydrase(29kDa).
F.DeLiseetal./JournalofMolecularCatalysisB:Enzymatic134(2016)136–147 139 2.7. Enzymeactivityassays
!-RHAactivitywasdeterminedusingpNPRassubstrate(pNPR assay). Otherwise stated, all activity assays were performed at RT. The reaction mixture contained, in a final volume of 0.5mLof50mMMOPSpH6.9,pNPRatafinalconcentrationof 600#M and variable amounts of the sample tested. The reac- tionwasblockedafter10 and20minbyadding0.5MNa2CO3;
the product, p-nitrophenolate(pNP), was detected spectropho- tometrically at $=405nm. The extinction coefficient used was %405=18.2mM− 1cm− 1.Oneunitofenzymeactivitywasdefined
astheamountoftheenzymethatreleasesonemicromoleofpNP permin.
2.7.1. Kineticparametersdetermination
KineticparameterswereobtainedatpH6.9usingapNPRcon- centrationintherange0.025–1mM.Allkineticparameterswere determinedbyanon-linearregressioncurveusingGraphPadPrism (GraphPadSoftware;www.graphpad.com).
2.7.2. pHoptimum
pHoptimumfor!-RHAactivitywasdeterminedintherange 4.7–8.8.Enzymeassayswereperformedasdescribedabove,using the following buffers: 50mM potassium acetate (pH 4.7–5.7), 50mMMOPS/NaOH(pH5.7–7.7)and50mMTris/HCl(pH7.7–8.8). 2.7.3. Temperatureoptimumandstability
Optimumtemperaturewasevaluatedbyperformingthestan- dardpNPRassayandincubatingthereactionmixtureatdifferent temperatures,intherange25–55◦C.
Thethermalstabilityoftheenzymewasdeterminedbyincubat- ingtheenzymeforoneand3hat30,40,50,60◦Candmeasuring,
aftereachincubation,theresidualspecificactivity. 2.7.4. Organicsolventstolerance
The tolerance of the enzymatic activity to the presence of organicsolventsinthereactionmixture,suchasDMSO,acetone orethanol,wasevaluatedbyperformingthestandardpNPRassay in50mMMOPSpH6.9towhicheither10%or50%ofsolventwas added.
2.8. Substratespecificity
Reactionswerecarriedoutin0.6mLof50mMNa-phosphate bufferpH7.0undermagneticstirringat40◦Cinthepresenceof
20mM of eitherarylglycoside and 0.25UofrRHA-P. Reactions weremonitoredovertime(0–24h)byTLCanalysis(systemsol- vent:EtOAc:MeOH:H2O70:20:10).CompoundsonTLCplateswere
visualizedunderUVlightorcharringwith!-naphtholreagent. Hydrolysisreactionsofmaltose,pullulan,starch,amylopectin, sucrose,raffinose,lactose,xylanfrombirchwood,xylanfromoat spelt,hyaluronicacid,!−cellulose,cellobiose,chitosan,"-Glucan from barley, laminarin, curdlan, fucoidan from Fucus versiculo- sus,rhamnogalacturonan,rutinosewereperformedusing2.5mg ofeachsubstrate,whichwassuspendedin0.5mLof50mMNa- phosphatebufferpH7.0.Thereactionwascarriedoutat40◦Cunder
magnetic stirring,in the presenceof 0.25Uof rRHA-P. Hydrol- ysis productswere monitored by TLCanalysis (system solvent HCOOH:HAc:H2O:2-propanol:EtOAc:1:10:15:5:25).
Flavonoidiccompoundssuchasnaringin,rutin,neohesperidin dihydrochalcone,werescreenedaspossiblesubstrates.Inthiscase, a6mMsolutionofeachcompoundinafinal volumeof1mLof 50mMNa-phosphatebufferpH7wasincubatedat40◦Cin the
presenceof0.25UofrRHA-P.Reactionswerecheckedovertime byTLCanalysis(t=0,15′,30′,60′,90′,120′,150′,180′,24h)with
thefollowingsolventsystem:EtOAc:MeOH:H2O70:20:10.Anaddi-
tionalhydrolysisreactionofnaringinwasperformedinconditions similartothosereportedaboveinthepresenceof10%DMSO,and