alpha-Rhamnosidase activity in the marine isolate Novosphingobium sp. PP1Y and its use in the bioconversion of flavonoids

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ContentslistsavailableatScienceDirect

Journal

of

Molecular

Catalysis

B:

Enzymatic

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

␣-Rhamnosidase

activity

in

the

marine

isolate

Novosphingobium

sp.

PP1Y

and

its

use

in

the

bioconversion

of

ﬂavonoids

Viviana

Izzo

a,c

_,

_Pietro

_Tedesco

a

_,

_Eugenio

_Notomista

a

_,

_Eduardo

_Pagnotta

b

_,

Alberto

Di

Donato

a

_,

_Antonio

_Trincone

b

_,

_Annabella

_Tramice

b,∗

a_Dipartimento_di_Biologia,_Università_di_Napoli_Federico_II,_Complesso_{Universitario}_di_Monte_S._Angelo,_Via_Cinthia_4,₈₀₁₂₆_Naples,_Italy b_Istituto_di_Chimica_{Biomolecolare,}_Consiglio_Nazionale_delle_Ricerche,_Via_Campi_Flegrei_34,₈₀₀₇₂_Pozzuoli,_Naples,_Italy

c_Dipartimento_di_Medicina_e_Chirurgia,_Università_degli_Studi_di_Salerno,_Via_S._Allende,₈₄₀₈₁_Baronissi,_Salerno,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received3January2014

Receivedinrevisedform6March2014 Accepted5April2014

Availableonline18April2014 Keywords:

␣-l-rhamnosidase Novosphingobiumsp.PP1Y Flavonoidsbioconversion

a

b

s

t

r

a

c

t

CrudeproteinextractsofNovosphingobiumsp.PP1Y,amicroorganismisolatedfrompollutedmarine watersinPozzuoli(Italy),wereanalyzedforthepresenceofglycosidaseactivities.Particularattention wasdevotedtoa␣-L-rhamnosidaseactivityabletohydrolyzeseveralﬂavonoidsofinterestforthe phar-maceuticalandfoodindustries.ThisactivityhadanalkalinepHoptimumandamoderatetolerancetothe presenceoforganicsolvents,appealingfeaturesforitspossiblebiotechnologicaluses.Anincreaseofthe ␣-L-rhamnosidaseactivityinPP1Ycrudeextractswasinducedbyaddingnaringintothegrowthmedium, suggestingthepossibilitytousematerialfromCitrusindustrialwastetoinducetheglycosidaseactivity expressedbystrainPP1Yandproducesimultaneouslyhigh-added-valuemoleculesfromthehydrolysisof theirﬂavonoids.InordertoinvestigateontheenzymaticmechanismofPP1Y␣-L-rhamnosidaseactivity, hydrolysisproductsofPNP-␣-L-rhamnopyranosidewereanalyzedby1_H-NMR_experiments._The_kinetic

1. Introduction

Biocatalysisrepresentsnowadaysaversatileandvaluabletool forindustrialbiotechnologies.Theuseofenzymesasbiocatalysts canhavesignificantbenefitscomparedtoconventionalchemical technologies,forachievinghighreactionselectivity,higher reac-tionrate,improvedproductpurity,andasignificantdecreasein chemicalwasteproduction.Awidevarietyofchemicalsubstances is already produced in industrial processes through theuse of enzymes[1,2].

Withinthisframework,␣-l-rhamnosidases[E.C.3.2.1.40]have attractedgreatattentioninthelastdecadeduetotheirapplication asbiocatalystsinavarietyoffood,pharmaceuticalandchemical industrialprocesses[3].

␣-l-Rhamnosidases(fromnowonindicatedas␣-RHAs)area classofglycosylhydrolasesthatspeciﬁcallycleaveterminal ␣-l-rhamnosefromalargenumberofnaturalproducts,whichinclude ﬂavonoids,someterpenylglycosides[4,5]andmanyothernatural glycosidescontainingaterminalrhamnose,suchasglycopeptides antibioticsandglycolipids[6].Theseenzymeshaverecentlybeen

∗ Correspondingauthor.Tel.:+390818675070;fax:+390818041770. E-mailaddress:atramice@icb.cnr.it(A.Tramice).

thefocusofanincreasingscientificinterest.Asanexample,theuse of␣-RHAstoimprovethebioavailability,andthusthebiological activity,offlavonoidsbeneficialforhumanhealthasdirectdrugs orasnutritionalsupplements,hasbeenreported[3].

Moreover,l-rhamnosehasacentralroleintheorganicsynthesis asachiralintermediateofpharmaceuticallyimportantcompounds. Itsproductionusingtheenzymaticactivityof␣-RHAsinhydrolysis reactionsofglycosylatedcompoundsthatmightberecoveredfrom wastematerialoffoodprocessingindustry(e.g.citruspeel), repre-sentsaninterestingandusefulbiotechnologicalapplicationthese glycosidases[7].

Fungiarethemainsourceof␣-RHAs.However,theseenzymes havebeenisolatedalsofromanimaltissues,suchastheliverofboth themarinegastropodTurbocornutus[8]andpig[9],andfromplants suchasRhamnusdaurica[10]andFagopyrumesculentum[11].

Bacteriarepresentayetunexploredreservoirof␣-RHAs,which mightshownovelinterestingproperties.Ananalysisofseveral ␣-RHAsisolatedfromvariousmicrobialsourcesreveals,forexample, thatoneofthemaindifferencesfoundbetweenfungaland bacte-rialenzymesistheiroptimalpH,withthefungalenzymesshowing more acidic pHoptimacompared tobacterialcounterparts, for whichneutralandalkalineoptimalpHvalueshavegenerallybeen reported.Thischaracteristicsuggestsdifferentpotential applica-tionsforfungalandbacterialenzymes,makingbacterial_␣-RHAs

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suitableinbiotechnologicalprocessesrequiring goodactivityin more basicsolutions such as theproduction of l-rhamnose by hydrolysisofnaringinorhesperidin,compoundswhosesolubility stronglyincreasesathigherpHvalues[3,12].

Despitetheverylimitednumberofbacterial␣-RHAsthathas beendescribedsofar,datainliteraturesuggestthatthisenzymatic activityis widelydistributed over a diverserangeof ecological niches.␣-RHAshavebeendetectedandcharacterizedinthehuman intestinalbacteriumBacteroidesJY-6[13],incold-adapted Pseu-doalteromonasspecies and Ralstoniapickettii isolatedin thesea waterofsubAntarcticenvironment[14],alsoinsoilbacteriasuchas Bacillussp.GL1[15,16],SphingomonaspaucimobilisFP2001[17]and Sphingomonassp.R1[18]andﬁnallyinwinestrainsofOenococcus oeni[19].

␣-RHAsfromLactobacillusspecieswereidentiﬁedand investi-gatedfortheirpotentialbiotechnologicalusetode-rhamnosylate ﬂavonoidspresentinfrequentlyconsumedfoodcommodities[20]. Moreover,␣-RHAsgeneshavebeenclonedandexpressedfrom thermophilic bacteriasuch asClostridiumstercorarium [21] and fromthebacteriumPRI-1686,whichisamemberofthephylum ofThermomicrobia[22].

Recently, the crystal structure of Streptomyces avermitilis ␣-l-rhamnosidase(SaCBM67)wasreported[23];itsstructure pre-sentedanovelcatalyticcarbohydrate-bindingmoduleandresulted different from the only two structures of ␣-l-rhamnosidases (GH78)previouslyreported:theBsRhaBisolatedfromBacillussp. GL1[24] and theputative␣-l-rhamnosidase BT1001from Bac-teroidesthetaiotaomicronVPI-5482[25].

Itgoeswithoutsayingthatthebiotechnologicalpotentialof bac-terial␣-RHAs,whosestructural,functionalandmolecularbiology aspectshavenotbeensufﬁcientlyinvestigated,isstrictlyrelated totheacquisitionofnewinformationontheenzymaticsystems isolatedfromnewbacterialsources.

Recently, Novosphingobium sp. PP1Y, an organic solvent-resistant, bioﬁlm-forming marine microorganism, was isolated fromthesurfacewatersofadockingbayintheharbourof Poz-zuoli(Naples,Italy),anareaheavilypollutedwithhydrocarbons

[26].Theanalysisofitsgenome,conﬁrmedthepresenceofseveral genomicfeaturesofinterestforthebiotechnologicalpotentialof thismicroorganism,andmorespeciﬁcallyforitsexploitationasa sourceofenzymesactiveoncarbohydrates.StrainPP1Yshowsin factauniqueabundanceamongSphingomonadalesofgenes encod-ingforglycosyl hydrolases(53orfs)[27],whichare distributed among27differentfamilies.Thispromptedourinterestin investi-gatingthepresenceof␣-RHAactivitiesinthecrudeproteinextract ofstrainPP1Y.

Inthiswork,cellextractsobtainedfromNovosphingobiumsp. PP1Ycellsgrowninminimalmediumweretestedforthepresence ofglycosidase activities;in particular␣-RHAand ␤-glucosidase activitiesweredetectedandpartiallycharacterized.

This ␣-RHA activity was successfully used in bioconversion studiesofﬂavonoidiccompounds,thussuggestingitspotentialuse asaneco-friendlytooltomodulatethebiologicaland pharmacolog-icalpropertiesofthesemolecules.Itwasfurtherdemonstratedby

1_H-NMR_experiments_that_␣-RHA_activity_from_{Novosphingobium}

sp.PP1Yfunctionedwithaninvertingmechanism,inagreement withtheenzymaticmechanismalreadyproposedforother␣-RHAs previouslydescribed.

2. Materialsandmethods

2.1. General

Novosphingobiumsp.PP1Ywasisolatedaspreviouslyreported

[26].AllreagentswerepurchasedfromSigma-Aldrichandused

withoutany furtherpuriﬁcation. Silica gel, reverse-phasesilica gelC-18andTLCsilicagelplateswerefromE.Merck(Darmstadt, Germany).Allotherchemicalswereofanalyticalgrade.Compounds onTLCplateswerevisualizedunderUVlightorcharringwith ␣-naphtholreagent.

TLCsolventsystemsusedwere(A)EtOAc:MeOH:H2O,70:20:10

and(B)EtOAc:AcOH:2-Propanol:HCOOH:H2O,25:10:5:1:15. 1_H-,13_C-_and_2D-NMR_spectra_were_acquired_by_the_NMR_Service

oftheIstitutodiChimicaBiomolecolareoftheNationalResearch CouncilofItaly(C.N.R.-Pozzuoli,Naples,Italy)andrecordedona BrukerDRX-600spectrometer,equippedwithaTCICryoProbeTM_,

ﬁtted with a gradient along the Z-axis, and on other Bruker instrumentswithﬁeldsat400and/or300MHz.SamplesforNMR analysisweredissolvedintheappropriatesolvent;spectrainD2O

were referenced to internal sodium

3-(trimethyl-silyl)-(2,2,3,3-2_H

4) propionate (Aldrich, Milwaukee, WI); for other solvents

downﬁeldshiftofthesignalofthesolventwasusedasinternal standard.

Inthebioconversionstudiesfollowedby1_H-NMR_experiments,

reportedin paragraph2.8 and 2.9,the selectedsignals integral valuesofproductsand reagentsresultedaffectedbyanerrorin integration(<5%)whichdependeduponinstrumentoptimization. Proteinconcentrationwasroutinelyestimatedusingthe Bio-Rad Protein System [28]; bovine serum albumin was used as standard.

2.2. Bacterialgrowth

Novosphingobium sp. PP1Y wasroutinely grown in minimal medium. Potassium phosphate minimal medium (PPMM) con-tained 20mM potassium phosphate pH 6.9, 1g/L NH4Cl and

100mMNaCl.Afterautoclaving,5mLofatraceelementsolution wasaddedtoeachlitreofcooledPPMM.Thetraceelement solu-tioncontainedina0.92%solutionofHCl:30.1g/LMgSO4,4.75g/L

FeSO4×7H2O, 5.4g/L MgO, 1.0g/L CaCO3, 0.72g/L ZnSO4×7

H2O, 0.56g/L MnSO4×H2O, 0.125g/L CuSO4×5 H2O, 0.14g/L

CoSO4×7H2O,0.03g/LH3BO3,0.004g/LNiCl2×6H2O,0.006g/L

Na2MoO4×2H2O. When usingPPMM as growthmedium, 0.4%

(w/v)of glutamic acid, preparedin deionized water and steril-izedbyﬁltrationwitha0.22␮mMilliporemembrane,wasused asuniquecarbonandenergysourceandaddedtotheautoclaved media. Cultures were incubated at 30◦C with orbital shaking (220rpm).

Bacterial growth was monitored by measuring the optical densityat600nm(OD600).Apre-inoculuminrichmediumwas

pre-paredbytransferring50␮Lfromaglycerolstockstoredat−80◦_C

toa50mLFalcontubecontaining12.5mLofsterileLBmedium.LB waspreparedaccordingtoSambrooketal.[29].Thepre-inoculum wasallowedtogrowat30◦Cforatleast14hat220rpmandthen usedtoinoculate1LofapreparativegrowthinPPMMmediumat aninitialcellconcentrationof0.01–0.02OD600/mL.Cellswere

usu-allyharvestedafter24hataﬁnalopticaldensityofca.1OD600/mL.

Itisworthytonotethat,whenfollowingthegrowthformorethan 24h,theformationofﬂockswasobserved,thushamperingacorrect estimationoftheturbidityofthecellsuspension.

2.3. PreparationofNovosphingobiumsp.PP1Ycrudeprotein extract

Cellcultureswereharvestedbycentrifugationat5,524×gfor 30minat4◦C.Cellpelletsweresuspendedin25mMMOPSatpH 7.0anddisruptedbysonication(30total,30sONand30sOFF)in anice-waterbath.Cellsuspensionwascentrifugedat17,418×gat 4◦Cfor60min.Thesolublefractionobtainedaftercentrifugation wasﬁlteredona0.45mmMilliporemembrane,dividedinaliquots andstoredat−80◦_C._When_used_for_the_screening_of_glycosidase

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Table1

Evaluationofdifferentsubstratesbioconversionbyusingglycosidaseactivitiesin thecrudeextractofNovosphingobiumsp.PP1Ycells.

Substratesa _Crude_extract_of_PP1Y_cells_grown_in_PPMM_medium Hydrolysisb _{Transglycosylation}c 3h 24h 3h 24h PNP-␣-d-Glcp + + − +/− PNP-␤-d-Glcp + ++ + ++ PNP-␣-d-Galp +/− + − +/− PNP-␤-d-Galp + ++ + ++ PNP-␣-d-Manp − − − − PNP-␤-d-Manp − − − − PNP-␣-d-Xylp − − − − PNP-␤-d-Xylp + ++ +/− + PNP-␣-l-Fucp − − − − PNP-␤-d-Fucp + ++ + ++ PNP␣-d-NAGp − − − − PNP␤-d-NAGp +/− + +/− +/− PNP-␣-l-Rhap ++ +++ − +/− PNP-␣-l-Araf − − − −

Note:−/+:percentageofproductsbelow10%;+:10–30%ofproducts;++:30–70%of products;+++:percentageofproductshigherthan70%.

a_Araf,_{arabinofuranose;}_Fucp,_{fucopyranose;}_Galp,_{galactopyranose;}_Glcp,

glu-copyranose;Manp,mannopyranose;NAGp,N-acetylglucosaminopyranose;Rhap, Rhamnopyranose;Xylp,xylopyranose.

b_Hydrolysis_products_were_analyzed_by_comparing_Rf_values_with_appropriate

standardsinTLCsolventsystemA.

c _Due _to _{self-transglycosylation} _reactions, _products _showed _positive _UV

absorbanceandlowerRfthanthecorrespondingarylsubstratesintheselectedTLC solventsystemA.

activities(Section2.4),thesolublefractionwasdialyzedagainst 20mMpotassiumphosphateatpH7,andthendividedinaliquots andstoredat−80◦_C._PP1Y_crude_extracts_were_assayed_for_their

totalproteincontent.

Withoutanydifferentindication,allexperimentsreportedinthe followingsections,wereperformedusingacrudeextractobtained froma2LcultureofPP1YcellsgrowninPPMMmediumandwith a proteinconcentrationof 3.72mgtotal protein/mL. Cellswere recoveredafter24hatanopticaldensityof1.02OD600/mL.

2.4. Glycosidaseactivitiesscreening

PP1Ycrudeproteinextract,preparedasdescribedinthe previ-ousparagraph,wastestedforthepresenceofglycosidaseactivities byusingseveralpNP-␣-andpNP-␤-substrates(Table1)ata20mM concentrationin0.6mLof50mMNa-phosphatebufferpH7,at 35◦Candundermagneticstirring.Allreactionswerecarriedout using4.42mgof totalprotein/mmol ofreagent. Reactionswere monitoredovertime(0–24h)byTLCanalysis(systemsolventA).

Moreover,hydrolysisreactionswithmaltose,lactose,cellobiose, sucrose,rafﬁnose,laminaripentaose,laminarin,curdlan,␤-glucan frombarley,xylan frombirchwood, pullulan, amylopectin and starchassubstrateswerecarriedoutusingcrudeextractsofPP1Y cellsgrowninPPMM.Eachsubstrate(10mg)wassuspendedin 1mLof50mMNa-phosphatebufferpH7,containing41.6␮gof totalprotein/mLofreactionmixture.Reactionswereperformedat 35◦C.HydrolysisproductsweremonitoredbyTLCanalysis(system solventsAandB).

Flavonoidicsubstratessuch asnaringin, diosmin, rutin, hes-peridin,neohesperidindihydrochalcone,quericitrin,weretested. Tothispurpose,a2mMsolutionofeachcompoundpreparedin aﬁnalvolumeof1mLof50mMNa-phosphatebufferpH7was incubatedat35◦Cinthepresenceof100␮LofPP1Ycrudeextract obtainedfromcellsgrowninPPMM(3.72mgoftotalprotein/mL) for24h.ReactionswerecheckedovertimebyTLCanalysis(solvent systemA).

Rutinosehydrolysiswasalsoinvestigated.Inthiscase,0.5mLof asolutioncontaining6mMrutinose,0.25mLofPP1Ycrudeextract in50mMofTris/HClbufferatpH8.5wasincubatedat35◦Cunder magneticstirringandmonitoredbyTLCanalysis(solventsystem A)for24h.

Inallexperiments,TLCstandardsolutionsofpurereagentsand productswereusedforcomparison.

2.5. _{˛-l-rhamnosidase}activityassay

␣-RHA activity was determined using p-nitrophenyl- ␣-l-rhamnopyranoside(pNPR)assubstrate.Theassaywasperformed in0.5mLof50mMpotassiumacetatebufferpH5.5,containinga variableamountofPP1YcrudeextractandpNPRataﬁnal concen-trationof0.28mM.Theassaymixturewasincubatedfor30min atroomtemperature;afterwards,0.5mLofa1Msodium carbon-atesolutionwereaddedandthesampleabsorbancewasrecorded spectrophotometricallyat405nm(ε405=0.0182␮M−1cm−1).One

milliunit of enzymatic activity was deﬁned as the amount of enzymethatreleases1nmolofp-nitrophenolpermin.

2.6. Inductionof_{˛-l-rhamnosidase}activity

Toevaluatethepossibleinfluenceofnaringin onthe expres-sionoftheintracellular␣-rhamnosidaseactivityofthestrainPP1Y, wecomparedthespecificactivityincellextractsobtainedfrom growthsinPPMMtowhichincreasingconcentrationsofnaringin wereadded.Tothispurpose,four250mL-Erlenmeyerflaskswere prepared,eachcontaining250mLofPPMMmediumand0.4% glu-tamicacidasuniquecarbonandenergysource.Variableamounts ofnaringinwereaddedtoautoclavedmedia,inordertohavefinal concentrationsof0.1–0.2–0.3mM.Thefourflaskswereinoculated withanovernightcultureofPP1YinLB,asdescribedinparagraph 2.2.Theinitialcellconcentrationsintheflaskswerecomparable andintherange0.02–0.04OD600/mL.

2.7. InﬂuenceofpHandorganicsolventson˛-l-rhamnosidase andˇ-glucosidaseactivities

InexperimentsofpHmonitoring,2mMsolutionsofnaringin werepreparedinHCl/KCl,Na-acetate,Na-phosphate,Tris/HCl, Na-carbonatebuffers,inordertocoverthepHrangebetween2.2and 10.9(Table2).100␮LofPP1Ycrudeextractwereaddedto0.5mL ofeachsolutionandincubatedat35◦Cundermagneticstirring. Reactionswerecheckedevery20minbyTLCanalyses(solvent sys-temA)andnaringinconsumptionwasevaluatedbyusingsolutions ofpurenaringin,narigenin,rhamnoseandglucoseas chromato-graphicstandards.BlankreactionsatdifferentpHswereperformed toevaluatethechemicaldegradationoftheﬂavanoneglycoside, occurringpreferentiallyinstronglybasicconditions.

Similarly,solutionsof2mMnaringinin50mMTris/HClbuffer pH8.5containing1,10and50%v/vofDMSOorCH3CN(Table2)

wereplacedunderconstantagitationat35◦Cinthepresenceof 200␮LofPP1Ycrudeenzymaticextract(0.74mgtotalprotein)/mL ofreaction.Aspreviouslyreported,reactionsweremonitoredby TLCanalysis(solventsystemA).

2.8. Stereochemicalcourseof˛-l-rhamnosidaseactivityin hydrolysisreactions

To the purpose, 800␮L of a 6mM pNPR solution prepared in 50mM Tris/HCl buffer pH7 was freeze-dried, re-suspended threetimesinD2Oandre-freeze-driedtoexchangethe1Hatoms

involvedinlabilelinkagesfor2_H_ones._A_similar_procedure_of_proton

isotopeexchangewasappliedto200␮LofPP1Ycrudeextract.Just priortothe1_H-NMR_experiment,_the_previously_exchanged_pNPR

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Table2

TheinﬂuenceofpHandorganicsolventspresenceonnaringinhydrolysisby␣-RHA activityinPP1Ycrudeproteinextract.

pHvalues Naringinhydrolysis Time(min) 60 120 140 180 2.2 − − − − 3.6 − − − − 5.4 − − − − 5.9 − − − −/+ 6.1 − −/+ −/+ + 7.2 −/+ + ++ ++ 8.1 − ++ ++ ++ 8.3 + ++ +++ +++ 8.5 + +++ +++ +++ 8.8 + ++ +++ +++ 9.1a ₋ ₋ ₊ ₊ 9.9a ₋ ₋ ₊ ₊ 10.9a ₋ ₋ ₊ ₊ %oforganicsolvent DMSO1% + ++ ++ +++ DMSO10% + + ++ +++ DMSO50% − − − − CH3CN1% − − − −/+ CH3CN10% − − − − CH3CN50% − − − −

Note:−nohydrolysis;−/+:percentageofhydrolysisbelow10%;+:10–30%of hydrol-ysis;++:30–70%ofhydrolysis;+++:percentageofhydrolysishigherthan70%.

a_In_these_conditions_a_partial_chemical_degradation_of_naringin_was_observed

whichwassuggestedbythepresenceonTLC,alongwiththedisappearanceofthe substrate,ofaproductmorepolarthannaringinandtheabsenceofrhamnoseand glucosespots,thusconﬁrmingtheabsenceofanenzymatichydrolysis.

solutionwasdissolvedin0.75mLofD2O,equilibratedat35◦Cina

NMRtubeinsidethespectrometerprobe,andaninitialspectrumof solutionwithoutenzymewasrecorded(T0).Thealiquotof

freeze-driedcrudeextractwassuspendedin50␮LofD2Oandaddedtothe

solution.Startingfromthatmoment(T0withenzyme),the

stereo-chemicalcourseofhydrolysiswasfollowedbyrecording1_H-NMR

spectraatcloseintervalsduringtheincubationtime (from0to 60min).

A6mMsolutionoffreerhamnosedissolvedin50mMTris/HCl bufferpH7wastreatedandmonitoredby1_H-NMR_experiments_as

previouslyreported.

2.9. Bioconversionstudiesfollowedby1_H-NMR_analyses

pNPR, naringin, rutin and neohesperidin dihydrochalcone bioconversionswereperformedandcomparedby1_H-NMR

spec-troscopicanalyses.

In particular,11.4␮mol of each substrate weredissolved in 1.9mLofNa-phosphatebuffer(50mM,pH8.5orpH7) contain-ing1mLofPP1Ycrudeextract.Reactionswereincubatedat35◦C, underconstantmagneticstirringfor3h.Aliquotsof380␮Lwere withdrawnfromeachreactionatdifferenttimeintervalsfrom0 to173min.Enzymaticreactionswerestoppedbyboilingat100◦C for2minandsampleswerefreeze-dried.Collectedaliquotswere dissolvedinMeODorD2O,asforpNPR,andanalyzedby1H-NMR

experiments.ThesereactionswerealsomonitoredbyTLC analy-sis(systemsolventA).ThesereactionswerealsomonitoredbyTLC analysis(systemsolventA).

2.10. Pruninproduction

Thereactionwasperformedusing2mLofPP1Ycrudeextract whichwerepreviouslylyophilizedandthenresuspendedina1mL solutionofNa-phosphatebuffer(50mM,pH8.5)containing10% ofDMSO.72.5mgofnaringin(0.125mmol)wereaddedandthe

samplewasincubatedat35◦C,undermagneticstirring.Reaction wasstoppedafter72hbyheatingthesampleat100◦Cfor2min andpruninwasisolatedbysilicagelpuriﬁcationwithEtOAc:MeOH 9:1(v/v)asmobilephase.1Dand2DNMRanalysesconﬁrmedits structure.

3. ResultsandDiscussion

3.1. Glycosidaseactivitiesscreening

Glycosidaseactivitiesincrudeproteinextractof Novosphingob-iumsp.PP1Ycellscollectedaftera24hgrowthinPPMMmedium (Section2.2)werepreliminaryinvestigatedfortheirhydrolyticand transglycosylationpotentials,asdescribedinSection2.4.Reaction productswereanalyzedovertimebyTLCanalysisusingsolvent systemASection2.1, andtheresultswerereportedinTable1. Dataindicatedthat␣-l-rhamnosidaseand␤-glycosylhydrolases activitiesresultedthemostabundantones.After3h,30–70%of PNP-_␣-l-Rhapand 10–30%of PNP-_␤-d-Glcp,PNP-_␤-d-Galp, and PNP-␤-d-Xylpwerehydrolyzed.Hydrolysisreactionswereallowed toproceedfor24h.Atthistime,analmostcompleteconversionof PNP-␣-l-Rhapwasobserved.

Transglycosylationreactionsresultedmoreinterestingbyusing PNP-␤-glycosides as substrates, suggesting a better attitude of ␤-glycosyl hydrolases to transfer monosaccharidic units in self-condensationreactionsthanthe␣-l-rhamnosidaseactivity; aryl-␤-oligosaccharideswerepresentinthereactionmediaupto 24h.

Polysaccharidesandoligosaccharideswerealsotestedas pos-siblesubstratesfortheenzymaticactivitiespresentinPP1Ycrude extracts.ProductsformationwasmonitoredbyTLCanalysis (sys-tem solvents A and B, Section 2.1), using pure compounds as chromatographic standards. After24hof reaction, curdlan and laminarinwereonlyslightlyconsumedwhereaslaminaripentaose resultedthebesthydrolyzedoligosaccharide,resultinginthemajor productionofglucose;cellobiose,instead,wasnotagoodsubstrate (datanotshown).

␣-glucanslikepullulan,amylopectin,starchandmaltosewere partiallyoralmosttotallyhydrolyzedtoglucose,maltoseorlarger oligosaccharides,suggestingthepossiblepresenceof␣-glucanase activities(datanotshown).

Thesepreliminarydataconﬁrmedthebiotechnological poten-tialofPP1Ystrainasasourceofawidearrayofenzymaticactivities thatcanbeusedforthemodiﬁcationofcarbohydratesand glyco-conjugates.

3.2. HydrolyticbehaviourofPP1Ycrudeextractenzymeson ﬂavonoids:description,pHdependenceandtolerancetoorganic solvents

ThesimultaneouspresenceinPP1Ycrudeextractof␣-RHAand ␤-glucosidaseactivitiespromptedustoinvestigatethe possibil-itytohydrolyzeselectedﬂavonoidiccompounds,havingintheir chemicalstructureboth␣-rhamnoseand␤-glucoseunits,suchas naringin,diosmin, rutin,hesperidin,neohesperidin dihydrochal-cone and quericitrin (Fig. 1).It is worth tonote that chemical modiﬁcationofthesecompounds,whichareendowedwith ther-apeuticpotential [12]is important tomodulatetheirbiological activity.

InthereactionconditionsreportedinSection2.4,after3hof incubation, complete conversionof naringin,rutin and neohes-peridin dihydrochalcone was detectedby TLCanalysis (system solventA,Fig.2):spotsofrhamnose,glucoseandthecorresponding aglyconewereobservedforeachreaction.

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Fig.1. Chemicalstructureofﬂavonoidiccompoundsusedassubstrates.Chemicalshiftvaluesofsignalsthatareintegratedinbioconversionreactionsarereported.

Ontheotherhand,diosmin,hesperidinand quericitrinwere nothydrolyzed;after24hasobservedbyTLCinvestigations (sys-temsolventA)rhamnose and glucosespots, atRf0.56 and 0.3 respectively,wereabsentonTLCplatesofdiosminandhesperidin

reactions.Quericitrinwassopoorlyconsumed(yieldbelow3%)that itshydrolysiswasconsiderednegligible;indeed,averyscarce pres-enceofquercetinandrhamnose,atRf0.88and0.56respectively, wasobserved.

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Fig.2.FlavonoidshydrolysisfollowedbyTLC(systemsolventA).TLCstandard solutionsataconcentrationof2mMwereused.Nar:naringin,R-nar:naringin reaction;rut:rutin,R-rut:rutinreaction;neohe:nehoesperidindihydrochalcone; R-neohe:nehoesperidindihydrochalconereaction;glu:glucose,rha:rhamnose;es: enzymaticsolution.

Thesedatagavesomeindicationonthesubstratepreferenceof the␣-RHAactivityexpressedbystrainPP1Yunderour experimen-talconditions.

Diosminandhesperidin(Fig.1),whichcontainedtherutinose disaccharide (␣-l-Rhamnopyranosyl-(1→6)-␤-d-glucopyranose) linked to the phenolic portion of the molecules, were not hydrolyzed. Rutin, whose rutinosideportion was linked tothe quercetin3-enol position,wasinsteadconverted. Undersimilar reaction conditions, ﬂavonoids with the neohesperidose disac-charide 2-O-(6-deoxy-␣-l-mannopyranosyl)-␤-d-glucopyranose) linkedtothephenolicportion(naringinandneohesperidin dihy-drochalcone)werecompletelytransformed.

These data suggested that the ␣-RHA activity was able to hydrolyzeboth␣,1-2and␣,1-6interglycosidiclinkages.Mostof the␣-RHAsreportedinliteraturearemainlyactiveon␣-1,2 gly-cosidiclinkages,toasmallerextenton␣-1,6linkages,andeven lessonotherglycosidicbonds[30].Interestingly,theenzymatic activityexpressedbystrainPP1Ywasspeciﬁcfor␣,1-2 intergly-cosidicbondswhenthedisaccharideunitofrutinosewaslinked tothephenolichydroxylgroups.Infact,diosminandhesperidin, whichsharedthepresenceofa␣,1-6interglycosidicbondinthe disaccharideunitslinkedtoaphenolicsite,werenothydrolyzed. The␣,1-6interglycosidicbondwasinsteadhydrolyzedwhenthe rutinoseunitwaslinkedto3-enolpositionofrutin.Thishydrolytic behaviourmightbeaconsequenceofadifferentstericeffect deriv-ingfromtheattachmentofthedisaccharidicunitstodifferentsites oftheaglycons[3].

Freerutinosedisaccharidewasalsoexaminedasapossible sub-strate,resultinginaverylowyieldofproducts(datanotshown), thus suggesting the importance of thearomatic portionof the ﬂavonoidicmoietyintherecognitionmechanismoftheenzymes involved.

Moreover,quercitrin,thequercetin3-␣-l-rhamnoside,inwhich l-rhamnoseisdirectlylinkedtotheaglycon,wasnothydrolyzed. ItshouldbenotedthatamarkedpreferencefortheofPNP- ␣-l-Rhap(fromnowonindicatedaspNPR)incomparisontoquercitrin, roboninorrutinhasbeenreportedalsoforothercharacterized ␣-RHAs[3].

The solubility of flavonoids (typically polyphenols) is pH-dependentand generallyincreasesinalkalinemedia,whichare oftennotcompatible withtheenzymatic activitiesusedin bio-processes. Using naringin as the most water-soluble available flavonoid(0.5g/Lat20◦C)[31],theinfluenceofpHandorganic sol-ventsonthehydrolyticbehaviouroftheenzymespresentinPP1Y crudeextractswasinvestigated.

ResultsoftheseexperimentswerereportedinTable2.Ahigher yieldofnaringinhydrolysisproductswasobtainedatalkalinepH valuesrangingfrom7.2to8.8(Na-phosphateandTris/HClbuffers) andanoptimumpHvalueof8.5wasdetermined.Inthiscase,after 3hofreaction,substrate spotswereabsentonTLCplates,thus suggestingasubstratecompletedepletion.

Withinareactiontimeof3h,TLCanalysesofreactionsystemsin thepHrange8.2–8.8showedthepresenceofaproductwithanRf valuehigherthanthatofnaringin.Freeglucoseandthenarigenin aglyconewerealmostabsent.

ThesedatasuggestedtheoccurrenceatthesebasicpHvaluesof amarkeddecreaseofthe␤-glucosidaseactivity,whichshouldbe responsiblefortheproductionoffreeglucoseandnarigenin agly-cone,andthepermanenceofthe␣-RHAactivity.Thishypothesis wasconfirmedbytheconcomitantaccumulationofacompound which wasidentified as prunin (Fig. 1), the de-rhamnosylated product ofnaringin, a moleculeof biotechnologicalimportance endowed with anti-inflammatory and antiviral activity against DNA/RNAviruses[32,33].

Itisworthtonotethatbacterial␣-RHAshavingapHoptimum valueat8–8.5havenotbeenoftenreportedinliterature[30].

Pruninstructure(Fig.1)wasconﬁrmedbya2DNMR spectro-scopicinvestigation.Thepresenceof the␤-glucoseresidue was establishedbythevalueoftheanomericpositionsignalin13_C_NMR

spectrum(inMeOD)at101.25ppmwhichresultedcorrelatedin HSQCspectrumtothediastereoisomericanomericprotonsH-1at 5.01(J=6.90Hz),and4.99(J=6.90Hz)ppm.Thefollowingsignals (inppm):C-274.66(H-2:3.48),C-377.82(H-3:3.47),C-471.16 (H-4:3.41),C-5 78.28(H-5:3.42),C-6 62.34(H-6-H-6:3.90, 3.72)conﬁrmedtheglucosestructureofpruninsaccharidicportion. Aglyconestructurewasconﬁrmedbythe13_C_spectrum_signals_at

80.69ppm(C-2),44.19ppm(C-3),166.98and167.05ppm(C-7,the glycosylationsite),129.12ppm(C-2-C6),116.33ppm(C-3-C5) andin1_H-NMR_spectrum_by_the_proton_signals_at_7.35_ppm

(H-2-H6)and6.85ppm(H-3-H5).Theothersignalsinthespectra wereinagreementwithdatapreviouslyreported[34].Theincrease ofthe␣-RHAactivitypresentinPP1Yproteinextractsobservedat alkalinepHvalueswasalsoconﬁrmedbythepNPRbioconversion studyfollowedbyNMRanalysisreportedinSection3.3.

ThepHvariationisnottheuniquestrategythathasbeenusedto overcomethepoorhydrosolubilityofﬂavonoids;theuseof water-miscibleorganicco-solvents,infact,hasalsobeensuggested[31]. However,itshouldbenotedthatthisstrategyhasseveral draw-backs,suchastoxicityproblemsderivedfromtheuseofanorganic solventinabioprocess,andthepossibledecreaseintheenzymatic activitiesemployed[35].

Inthisstudy,theinﬂuenceofthepresenceoforganicsolvents onthehydrolysis of naringinwas evaluatedby TLCanalysisof

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reactionmixturescontainingfrom1to50%v/vDMSOorCH3CN

(Table2).After90min,inthepresenceof1%and10%ofDMSO, naringinwasalmosttotallyconsumedandtracesofpruninwere detected,suggestingatoleranceofthe␣-RHAactivitytothe pres-enceoforganicsolventsalongwithadetrimentaleffectofDMSO onthe␤-glucosidaseactivity,asfreeglucosewasalmostabsentin thereactionmixtures.

Finally,inthepresenceofeither50%DMSOoranyconcentration ofCH3CN,de-rhamnosylationandde-glucosylationreactionsdid

notproceedevenafter10h.

Usingtheapparentbestexperimentalconditionsreportedin

Table2(pHvalueof8.5and10%DMSO),andstartingfroma125mM naringinsolution(Section2.10),pruninwasproducedwithayield of32.1%,correspondingtoaﬁnalconcentrationof0.02Mandan amountof17.4mgofrecoveredproduct.

Furthermore,free l-rhamnose wasproduced as a secondary productataconcentrationof6g/L.Theyieldofproductsobtained inthisexperimentwascomparabletothatreportedfora recombi-nant␣-RHAfromClostridiumstercorariumusedforthehydrolysis ofcitruspeelwastenaringin[7]andtoothersimilarprocesses[30]. 3.3. BioconversionexperimentsfollowedbyNMR

Toconﬁrmandbetterdetailtheobservationsonthesubstrate preference of the␣-RHA activityin Novosphingobium sp. PP1Y crudeproteinextractsdescribedinthepreviousparagraph, bio-conversionreactionsofpNPR,naringin,rutin andneohesperidin dihydrochalconewereperformedusingPP1Ycellextracts(Section

2.9),andmonitoredovertimeby1_H-NMR.

Basedondiagnosticsignalsofreagentsandproductsforeach reaction,substrateconversionswerecalculatedbymeasuringthe percentageratiobetweentheintegralsofdiagnosticsignalsofthe productsandthesumoftheintegrationvaluesofselectedsignals ofreagentsandﬁnalproducts.ResultswerereportedinFig.3.

The highest yield of bioconversion in a shortest time was obtainedwithrutin.After20min76.2%ofthesubstratewas con-sumed.Atthesametime,30–35%ofpNPR,26%ofnaringin,and7% ofneohesperidindihydrochalconeweretransformed.

After 173min, naringin and neohesperidin dihydrochalcone reachedbioconversionsyields(95.6and86%,respectively)similar torutin(89.4%).Undertheseconditions,3.1–3.3g/Lofﬂavonoids wereconverted toproductsand, although the ﬂavonoidic sub-strateswerenottotallyhydrolyzedtoaglycone,freel-rhamnose wasreleasedintothereactionmediaataconcentrationofabout 0.77–0.86g/L.

ItshouldbeaddedthatpNPRbioconversionwasperformednot onlyatpH8.5butalsoatpH7,lookingforapossiblepHinﬂuenceon PP1Y␣-RHAactivity(seeSection3.2).Intheexperimentinwhich naringinwasusedasasubstrate,thereactioncoursewas evalu-atedbycomparingin1_H-NMR_spectra_the_integral_values_of_two

aromaticprotonssinglets(Fig.1)H6–H8inringBat6.19ppmand 6.28ppm,whichwereattributedavalueof2,totheintegrationof thecorrespondingprotonsoftheﬁnalproductnarigenin(aglycon) at5.78and5.8ppm.1_H-NMR_spectra_in_MeOD_of_naringin,

narin-geninandprunin(H6–H8signalsat6.22–6.24,respectively)ofpure solutionsconﬁrmedtheidentityofnarigeninintegratedsignals.

Whenrutinwasusedassubstrate,aromaticprotonsignalH6 ofthereagentat6.18ppmwasselectedasreferenceforthe inte-gration(Fig.1).Asforthereactionproducts,anaromaticsignalat 6.13ppm,whichincreasedovertime,wasselectedforevaluating rutinbioconversion.

After10minofreaction,theanomericprotonof␤-glucosein rutinat5.07ppm(d,J=7.61Hz)almostdisappeared(72%of con-version)andananomericprotonsignalat5.035ppm(d,J=8.07Hz), possiblycorrespondingtothe␤-anomericprotonofquercetin3- ␤-glucoside,appeared;anomericsignalsoffreeglucosewereabsent.

On theotherhand,in1_H-NMR_spectra_at₁₇₃_min,_anomeric

protonsregionwasmorecrowdedwithsignalsduetothe pres-enceoffreeglucose(␣-protonat5.13ppmJ=3.91Hz;␤-protonat 4.50ppm,J=7.8Hz)andasignalat5.023ppmoffreerhamnose ␣-proton(withthe␤-anomericprotonoverlappedbythesolvent residualwatersignal).

Thesedatasuggestedafasthydrolysisoftheinterglycosidic link-ageinsidetherutinoseunit(_␣-l-Rha-(1_→6)-_␤-d-Glu)withthe productionoffreerhamnoseatthebeginningofthereaction,as conﬁrmedbythepresenceofafurthermethylsignalat1.27ppm after10minofreaction,correspondingtoprotonsatposition6of freerhamnose,andthesubsequentreleaseofﬂavonolquercetin andglucose.

TheNeohesperidin dihydrochalconebioconversionwas eval-uated by 1_H-NMR _using _the _aromatic _signals _at _6.049_ppm,

corresponding totwo overlapped aromaticprotons H2 and H6 (Fig.1)linkedtothesaccharidicportion,asthereagentreference signals,withaintegralvalueof1,andasignalat6.077ppm, belong-ingtothereactionproduct.

Anevaluationoftheanomericsignalsintherange4.45:5.2ppm of1_H-NMR_experiment_showed,_after₁₇₃_min_of_reaction,_the

pres-enceofthe␣-protonsignaloflinkedrhamnoseat5.28ppmandthe anomericsignalof ␤-glucoseat5.065ppm(d,J=7.63Hz)which belongedto thesubstrate, the␣- and ␤-anomericprotons sig-nalsoffreeglucoseat5.13ppm(d,J=3.81Hz)and4.50ppm(d, J=7.63Hz)respectively.Moreover,anintensesignalat5.03ppm ofthe␣-anomericprotonoffreerhamnose,asignalat4.95ppm (d,J=7.31Hz)almostasintenseastheprevioussignal,whichwas partiallycoveredbyresidualH2OsignalinMeOD,corresponding

to␤-glucoseH1signalofneohesperidindihydrochalconewithout rhamnosewerealsoobserved.Onthecontrary,thefreerhamnose ␤-anomericsignalwastotallyoverlappedbyresidualH2Osignalin

MeOD.

Thehighintensityofthe_␣-anomericprotonoffreerhamnose andthescarcepresenceofanomericsignalsoffreeglucose,ledusto supposethatthemainreactionproductwasthede-rhamnosylated neohesperidindihydrochalcone.

InexperimentsusingpNPRassubstrate,theintegralvalueof aromaticprotonsignalat7.23ppmwassetto1andthesignalof p-nitrophenolat6.69ppmproducedbythehydrolysisofglycosidic linkagewasselectedfor theassessment ofpNPRbioconversion. ThisbioconversionwasperformedattwopHvalues;asreportedin

Fig.2,after60min,theconversionofpNPRwasofabout61%atpH 7,whereasathigherpHitreached97%,thusconﬁrmingthetrendof theexperimentsinwhichnaringinwasusedassubstratedescribed inSection3.2.

3.4. Inductionof˛-RHAactivityinPP1Ycellextracts

␣-RHAactivitycanbespeciﬁcallyinducedbyseveralﬂavonoids

[36,37]amongwhichnaringinisofparticularinterestbecausethis compound can be abundantly recoveredin citrus solid wastes. Thesewastesarequiteinterestingbecausetheycanbeusedeither asanenergeticsourceforgrowingmicroorganisms,duetotheir content in carbon and othernutrient components,and/or as a speciﬁcinducerforthebiosynthesisofglycosidasesduetothe pres-enceofﬂavonoids.It goeswithoutsayingthattheuseofcitrus wasteasstartingmaterialinbioconversionprocessesisan advan-tageforindustrialcompaniesbecause,amongothers,itdecreases theexpensesforwastedisposal.Thepossibleroleofnaringinas an inducerof the␣-RHA activity[37] in PP1Y protein extracts wasinvestigatedusingPP1YcellsgrowninPPMMmedium sup-plementedwithnaringinatdifferentinitialconcentrationsupto 0.3mM,asdescribedindetailinSection2.6.

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Fig.3.Bioconversionsofnaringin䊉,rutin,neohesperidindihydrochalcone—,p-nitrophenyl-␣-l-rhamnopyranosideatpH7and8.5.

Theeffectofnaringincouldnotbeevaluatedatconcentrations higherthan0.3mMduetoitspoorsolubilityunderthe experimen-talconditionsused.

Theeffectofnaringincouldnotbeinvestigatedat concentra-tionvalueshigherthan0.3mMduetoitspoorsolubilityunderthe experimentalconditionsused.ResultswerereportedinFig.4and conﬁrmedthatnaringinactsasaninducerleading,ata concentra-tionof0.3mM,toamaximum5-foldincreaseofthe␣-RHAactivity (15.2mU/mg)detectedinthecrudeextractof PP1Ycells.These dataencourageafuturebiotechnologicaluseofPP1Ystrainandof its␣-RHAactivityfortheuseofagro-industryvegetableresidues. 3.5. Stereochemicalcourseof˛-RHAactivityinhydrolysis

reactions

Toshed light onthetype of enzymatic mechanism of PP1Y ␣-RHA activity,hydrolysis products of pNPRwere analyzed by recording 1_H-NMR _spectra _over _time. _The _integration _of _the

anomericprotonsignalsofreagentandproductswasinvestigated; theanalysiswasperformedtakingintoaccountthattheanomeric protonoffree rhamnoseshows peaksat5.06ppm (␣-anomeric form)and4.83ppm(␤-anomericform).Resultswerereportedin

Fig.5,wherethereactiontime-dependentchangesinamplitude ofH-1 signalsfrom substrate,␣-1(S) (5.68ppm),and products,

Fig.4.␣-RHAspeciﬁcactivityinthecellextractofstrainPP1Yisexpressedas mU/mgoftotalproteins(Y-axis).Speciﬁcactivityisreportedasafunctionofthe concentrationofnaringininitiallyaddedtothegrowthmedium.

␣-1(P),and␤-1(P),wereplottedas%oftotalanomeric(H-1)signals. Att0,theonly␣-anomericprotonsignalpresentwasthatofpNPR

at5.68ppm(␣-1(S)).Duringtheﬁrstminutesofreactionasignal at4.83ppm,correspondingtothe␤-l-Rhapanomericproton(␤-1 (P)),appearedonlyslightlyearlierthantheappearanceofafurther signalat5.06ppm,correspondingtothe␣-l-Rhapanomericproton (␣-1(P)).

These signals increased in intensity over time, while the anomericsignalofpNPRdecreasedconcomitantly,asitisevident fromFig.5.NMRanalysisdidnotgaveevidencefortheonsetof mutarotationequilibrationduringtheﬁrstminutesofreaction.

Laterintheincubation,mutarotationoftheinitiallyreleased ␤-l-Rhapincreasedtheratiobetweentheintensitiesofthe␣-and ␤-anomerprotonssignals.After7min,ataconversionpercentage of7.5%,therelativeintensitiesofthe␣-and␤-anomerresonances (calculatedfrompeaksintegration)wereina ratioofabout1:3 (25%ofthe␣-and75%forthe␤-anomer).After20min,ata con-versionpercentageof29%,the␣-/␤-anomerratiowasofabout1:2 (32%ofthe␣-and68%forthe␤-anomer).After60min,ata con-versionpercentageof61.5%,the␣/␤ratiovalueincreasedtoabout 1:1.Thisratiowaskeptconstantalsoafter80min,whenthe inte-grationvaluesshowedthattheyieldofpNPRconsumptionwasof

0 10 20 30 40 50 60 70 80 90 100 2 7 10 12 20 40 60 80 pe rcentage % time (min)

PNP-αα- Rha hydrolysis

Fig.5. %oftotalanomericsignalsinp-nitrophenyl-␣-l-rhamnopyranoside hydrol-ysisreaction:signalsfromsubstrate,␣-1(S),andproducts,␣-1(P),and␤-1(P) ,areplottedas%oftotalanomeric(H-1)signals.

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about86%,andwasmaintainedupto18h,thusrepresentingthe equilibriumvalueoftheratiobetweenbothanomericformsof l-rhamnopyranose.Asolutionoffreerhamnoseusedin thesame experimentalconditionsandmonitoredby1_H-NMR_reached_the

mutarotationalequilibriumafter1–2minandtheconcentrations ratioof␣-and␤-rhamnosewas1:1.

ThekineticbehavioursuggestedbytheseexperimentsforPP1Y ␣-RHAactivitywasaninvertingmechanismofhydrolysisinwhich ␤-rhamnosewasformedfromthe_␣-rhamnoseviaa single dis-placementmechanismandwasthenspontaneouslyconvertedby mutarotationtothe␣-anomericform.

4. Conclusions

Bacterialglycosylhydrolasesarethefocusofanincreasing num-berofresearchesbecauseoftheirkeyroleinfundamentalbiological processesandtheirbiotechnologicalapplications.Here,the atten-tionwasfocusedonanovel␣-RHAactivityfromNovosphingobium sp.PP1Y,whichresultedinterestingforitsalkalinepHoptimum andmoderatetolerancetoorganicsolvents.

This␣-RHAwasused,evenwithoutanyfurtherpuriﬁcationof PP1Ycrudeproteinextract,forthebioconversionofﬂavonoids use-fulforthefoodandpharmaceuticalindustries.However,afuture detailedbiochemicalcharacterizationofthisenzymeisnow cru-cial for a better understanding of its effective biotechnological potential.

Acknowledgments

WethankD.Melck,E.CastelluccioandA.Esposito(NMRservice ofIstitutodiChimicaBiomolecolare,CNR,Pozzuoli,Italy)fortheir skilfulassistance.

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