Current Research in Microbiology - Open Access. eBooks · Current Research in Microbiology 5 to be optimally active at neutral pH and are stable over a wide pH range. The enzymes

Cholesterol Oxidases: Microbial Enzymes in Industrial Applications

Chapter 5

Current Research in Microbiology

Sunita Devi; Shamsher Singh Kanwar*

Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla-171 005, India.*Correspondence to: Shamsher Singh Kanwar, Department of Biotechnology, Himachal Pradesh University,

Summer Hill, Shimla-171 005, India

Phone: 91-177-2831948; Fax: 91-177-2832153; E-mail: [email protected]

Cholesteroloxidasesarebifunctionalflavoenzymesproducedbydiversebac-terialspecies.Theseenzymescatalysetheoxidationofsteroidsubstratescontainingahydroxylgroupatthe3β-positionofthesteroidringbackbone.Theenzymeexistsintwomainforms:onewiththeFADcofactorembeddednoncovalentlyintheen-zyme;andonewiththecofactorattachedcovalentlytotheprotein.Cholesteroloxi-daseisofsignificantimportanceowingtoitsuseinanalysisofcholesterolamountinvariousclinicalandfoodsamples.Inaddition,theenzymealsoactsasalarvicidebiocontolagentagainstmanyinsectsandisalsoinvolvedinthebiotransformationofanumberofsteroids.Moreover,cholesteroloxidaseisalsoassociatedwithsomebacterialinfectionsandthuscanbeexploredasapotentialnewantimicrobialdrugtargettocontainbacterialinfections.

1. Introduction

Thehistoryofcholesteroloxidasedatesbacktoover70yearswhensearchforcholes-terol-degradingmicroorganismswas startedandmanyspeciesofMycobacterium were ob-servedtousecholesterolasasourceofcarbonandenergy[1,2].Ineukaryotes,cholesterolisessentialformaintainingcellmembranestructureandforsynthesizinganumberofimportantcompounds.Cholesterol is either derived exogenously or synthesized endogenously in theendoplasmicreticulum[3].Anabnormallyhighlevelofcholesterolinthebloodleadstoheartdisease,inadditiontomanyotherseverehealthproblems.Highercholesterollevels(greaterthan 200mgdL-1)may cause hypertension, arteriosclerosis, lipidmetabolismdysfunction,brainvessel thrombosis, nephritis, diabetes, jaundiceandcancer.Alternatively, abnormallylowcholesterollevelsmaycausehyperthyroidism,anemiaandmalabsorption[4,5].Several

CurrentResearchinMicrobiology

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www.openaccessebooks.comKanwarSS

compoundssuchasstatins,filipinandcyclodextrinsmaybeusedtomanipulatecellularcho-lesterol [6].Cholesteroloxidase isabacterialenzyme thathasproven tobeveryuseful inbiotechnologicalapplicationsrelatedtothedetectionofcholesterolandtothedisruptionofcholesterol-containingmembranes.Cholesteroloxidaseiscommonlyusedtomanipulatecho-lesterollevelsofcells[7].

Cholesteroloxidasehasmanyapplicationsandoneofmostimportantoneisitswideuseforthedetectionofamountofcholesterolpresentinfood,serumandotherclinicalsamplesthroughanenzymaticassay[8,9].Withtheexceptionofglucoseoxidase,thisenzymehasbe-comeoneofthemostwidelyusedbiosensorsinclinicalapplications[10].Cholesteroloxidaseisalsousedfortheproductionofavarietyofintermediateswhichareprecursorsforthesynthe-sisofsteroidhormones,suchas4-androstene-3,17-dione(AD)and1,4-androstadiene-3,17-dione(ADD)[11].Cholesteroloxidaseplaysacriticalroleinsterolcatabolismbyconverting3-β-hydroxysteroidsto3-oxo-4-enesteroids[10,12,13].Cholestenoneformedbycatalysisofcholesterolbycholesteroloxidaseisalsoanimportantsyntheticintermediateinmanysteroidtransformations.Previousstudieshaveshownthatitiseffectiveagainstobesity,liverdiseaseandkeratinization[14].Additionally,theenzymehasinsecticidaleffectagainstlarvaeofsomeinsects[15],alsousedformembranestructureanalysisandhypothesisedtoactasasignallingmoleculeforthebiosynthesisofpolyenemacrolidepimaricin.

Cholesteroloxidase(3-βhydroxysteroloxidase,EC1.1.3.6)isamemberofflavinad-eninedinucleotide(FAD)dependentenzymessuperfamilyandcatalyzestheoxidationofcho-lesterol(cholest-5-en-3β-ol)toits3-keto-4-enederivative,cholestenone(cholest-4-en-3-one),withthereductionofoxygentohydrogenperoxide[16].However,somebacterialcholesteroloxidaseshavealsobeenreportedtocatalyseoxidationofcholesterolto6β-hydroperoxycholest-4-en-3-one(HCEO)inplaceofcholest-4-en-3-one(CEO)[18].Cholesteroloxidaseisabac-terialFAD-dependentenzymewhichisfoundtoexistintwodifferentformsonthebasisofbondingbetweenenzymeandFADcofactor.InfirstformtheenzymeanditsFADcofactorarelinkednon-covalently(classI)andinsecondformcofactorisboundcovalentlytotheenzyme(class II) [18,19]. Cholesterol oxidase is among thewell-studied enzymeswith prominentcharacteristics,structuralfeatures[20],broaderbiotechnologicalapplications[21]andphysi-ologicalfunctions[22,23].Nonetheless,reportsregardingcholesteroloxidaseproduction,pu-rification,molecular characterization and genetic analyses have been previously published[24].

Many bacteria belonging to the genera Brevibacterium [25], Streptomyces [26,27],Corynebacterium [28],Arthrobacter [29],Pseudomonas [30],Rhodococcus [31,32],Chro-mobacterium[17]andBacillus[33,34]arereportedtodegradecholesterol.Microbialcholes-terolbreakdownoccursbythedegradationofitsside-chainandtheringtoacetylCoAandpropionylCoAthroughamulti-stepprocessandtheenzymewhichcatalyzesthefirststepis


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cholesteroloxidase.Mostofsteroidsincludingcholesteroldonotseemtobeabletodiffusetothebacterialcytoplasm,andappearstobetransportedbyspecificuptakesystemsbeforebeingmetabolized.Inmostofcholesteroldegradingmicroorganisms,thecholesteroloxidaseisem-ployedintheinitialstepofcholesterolmetabolism,whileincaseofpathogenicbacteriaitactsasmembrane-damagingfactorcontributingasavirulencefactorandaddstothepathogenicityofthesebacteria[35,36].

Figure 1:Thereactionmechanismofcholesteroloxidase.

2. Sources of Cholesterol Oxidase

Cholesteroloxidasehasbeenisolatedfromvariousbacterialsources,comprisingbothGram-negativeandGram-positivebacteria [10,23].Cholesteroloxidaseproducingbacteriaproducesitinthreeforms:intracellular,extracellularandmembranebound.Mostofthemicro-organismsdescribedascholesteroloxidaseproducers,produceitinextracellularformwhichiseventuallyreleasedintothefermentationmedium.Thecell-surface-linkedformofenzymeisextractiblewithnon-ionicdetergentssuchasTritonX-100(polyethyleneglycoloctylphenylether)andLubrolPX(polyethyleneglycolmonododecylether)[37,38,39,40].Themostcom-monmicrobialsourcesforproductionofcholesteroloxidaseareArthrobacter[41,42],Bacillus [33,43],Brevibacterium[44], Bordetella[45],Corynebacterium[28], Mycobacterium[46,47],Nocardia [48,49],Rhodococcus [32,38]andStreptomyces species.[26,50,51].SomeGram-negativebacteriasuchasBurkholderia[52],Chromobacterium[17], Enterobacter [53]andPseudomonas species.[30]havealsobeenreportedtoproducecholesteroloxidase.

Cholesterol oxidase is amonomeric enzymewhich exists in two forms classified asclassIandclassII,inclassItheFADcofactorisattachednon-covalentlytotheproteinandinclassIIthiscofactorisboundcovalentlytotheenzyme[54,55].Howeverthesetwoclassesofenzymeshowsamecatalyticactivitybutdonothaveanysignificantsequencehomologyandthusappeartobelongtodifferentproteinfamilies[23].ClassIenzymesarepartoftheglucose-methanol-choline(GMC)oxido-reductasefamilyandhavebeenfoundmostlyinac-tinomycetessuchasStreptomyces, Brevibcterium, Rhodococcus, Arthrobacte, Nocardia and Mycobacteriumspp.Whereas,ClassIIenzymeshavebeenassignedtovanillylalcoholoxi-

CH3

CH3

CH3

CH3

H3C

CH3

CH3

CH3

CH3

H3C

CH3

CH3

CH3

CH3

H3C

CHOxFAD FAD+H+

CHOx

O2 H2O2

OH O

O

Cholesterol

4-Cholesten-3-one

5-Cholesten-3-oneOxidation

Isomerization


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dase(VAO)familyandhavealsobeenreportedfromBrevibacterium sterolicum, Rhodococcus erythropolisandsomeGram-negativebacteriasuchasBurkholderia sp.,Chromobacterium sp.andPseudomonas aeruginosa[56].Mostofflavoenzymescontainaconsensussequencemadeupofrepeatingglycineresidues(GXGXXG)continuedbythepresenceofasparticacid⁄glutamicacidapproximately20residuesfurtherisindicativeofanucleotide-bindingfold[57].Thenoncovalentformofcholesteroloxidaseshowsanearlyidenticalconsensussequenceofglycinemoieties(G17-X-G19-X-G21-G⁄A22)whichisfollowedbyaglutamateandconfirmsthepresenceofnucleotide-bindingfold.However,thesecondformwhichexhibitscovalentbindinglacksthisconsensussequencewhichindicatestheprobableabsenceofanucleotide-binding fold [20].Despite exhibiting structural differences both covalent and noncovalentenzymeformspossessaburiedhydrophobicpocketforbindingsteroidringbackbone.Basedonfunction,cholesteroloxidasecontainstwodomains,theFAD-bindingdomainandthesub-strate-bindingdomain.Thereisnosignificantsequenceidentitybetweentwotypesofenzymesresultingindifferenttertiarystructureandkineticsmechanism[20,58].Ithasbeenreportedfromdifferent structural andkinetics studies thatHis447andGlu361 residues act asmaincatalystsalongwiththeconservedwatermoleculeH2OandAsn485intypeIcholesteroloxi-dase[59].The3-OHgroupofthesteroidringislinkedtoboththeFADcofactorandaboundwatermoleculeviahydrogenbonding.ThecriticalresiduescomposingtheactivesiteoftypeIIcholesteroloxidasefromBrevibacterium sterolicumincludeArg447,Glu475,Glu311,andAsn516[54].

Cholesteroloxidaseisthusafunctionalflavoenzymewhichcatalyzestheoxidationandisomerizationofcholesterol to4-cholesten-3-one in three successivesteps (Figure 1).Thefirststepresultsinthe3-OHgroupdehydrogenationwiththelossofthe3α-hydroxyand3β-hydroxyfromthesteroidringbackbone(reductivehalf-reaction).ThetworesultingelectronequivalentsaretransferredtotheoxidizedFADenzymecofactorthatisthenceconvertedtoitsreducedformintheprocess.Inthenextstep,thereducedformofFADcofactorreactswithmolecularoxygen(O2)toregenerateoriginalenzymeinitsoxidizedformandH2O2(oxidativehalf-reaction).Inthefinalthirdstep,thecholesteroloxidasecatalyzesisomerizationofdoublebondinthesteroidringbackbone,from∆5-6to∆4-5,leadingtofinalproductformation.IthasalsobeenreportedpreviouslythatcholesteroloxidasefromB. cepacia, Pseudomonasspp.,and Chromobacteriumsp.oxidizescholesteroltoHCEO[17,60]TheHCEOformationschemediffersonlyforasinglestepinwhichHCEOisformedfromcholest-5-en-3-one,presumablybyauto-peroxidation[23].

3. Properties of Cholesterol Oxidase

Cholesterol oxidases are amongwell studied enzymes and have been reported fromseveralmicroorganisms.Generalrangeformolecularweight(Mr)ofcholesteroloxidaseisre-portedtobefrom47–61kDa.Cholesteroloxidasesfromdifferentmicrobialsourcesarefound


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tobeoptimallyactiveatneutralpHandarestableoverawidepHrange.Theenzymeshavetemperatureoptimaintherangeof37–60°C(Table 1).CholesteroloxidasefromStreptomyces fradiaeisreportedtohavehighestoptimumtemperatureof(70°C)amongtheenzymesreport-ed[51].CholesteroloxidaseproducedfromChromobacteriumsp.strainDS-1isfoundhighlythermostableretaining80%ofitsoriginalactivityevenat85°Cafter30min[17].Generallycholesteroloxidasedoesnotrequiremetalionsforitsactivitybutincaseofsomeenzymes,itsactivitywasenhancedinthepresenceofmetalions[45,32].DifferentchelatingagentslikeEDTA,o-phenanthroline,and8-hydroxyquinolinealsodonotseemtohaveanysignificantef-fectontheenzymeactivity[17,61].Sincecholesterolisaninsolublecompound,detergentsaswellasorganicsolventsareoftenaddedtothereactionsolutiontoactassolubilizer(s).Cho-lesteroloxidasehasbeenusedfortheopticalresolutionofnon-steroidalcompounds,allylicalcohols[62,63]andthebioconversionofanumberof3β-hydroxysteroidsinthepresenceoforganicsolvents[64,65].Therefore,anorganicsolvent-tolerantcholesteroloxidasewouldbeusefulfortheseapplications.Organicsolventsofteninfluencethecholesteroloxidaseactivity[49,66].Table 1:Propertiesofcholesteroloxidasesproducedfromdifferentmicrobialsources.

Producer organism

Optimum pH

OptimumTemp. (°C)

Mr(kDa)

Temp. stability (°C)

Km(mM)

Specific activity (Units/mg)

References

Brevibacterium sterolicum

6.5 55 46.5 - 0.03 55.2 [44]

Streptomyces aegyptia NEAE

1027.0 37 46 50 0.152 16.1 [27]

Bacillus subtilis 7.5 37 105 37 3.25 1.39 [33]

Bordetella sp. 7.0 37 55 50 0.556 20.8 [45]

Chryseobacterium gleum

6.75 35 60 - 0.50 15.5 [67]

Rhodococcus sp. 8.0 37 60 - - 35.6 [32]

Enterococcus hirae 7.8 40 60 92 - 124.9

Streptomyces sp. 7.2 50 55 65 0.02 20.0 [26]

Chromobacterium sp. DS-1

7.0 65 58 85 0.026. 13.9 [17]

Burkholderia cepaca ST-200

6.8-8.0 60 60 70 - 16.9 [52]

Pseudomonas aeruginosa

7.0 70 59 70 92.6 11.6 [68]


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4. Substrates for Cholesterol Oxidase

CholesteroloxidaseisactiveonalargenumberofsterolswithorwithoutaC-17alkylchain.However, it ismostactiveinthepresenceofcholesterol.Richmond(1973)reportedthatcholesteroloxidasefromaNocardiasp.wasaffectedlargelybythelengthofsidechain[48].Longsidechainsappearedtoaidtheorientationofthesubstratewithrespecttotheac-tiveenzymaticsite.Allainetal., (1974) found thatergosterol,5,7-cholestadien-3β-ol,20α-hydroxycholesterol, 5α-cholestan-3β-ol and 7-cholesten-3β-olwere oxidised at rates lowerthanthatoftherateofcholesterolforthesameenzyme[69].IncontrasttotheenzymefromNocardiasp.,acholesteroloxidasefromBrevibacterium sterolicumwasfairlyreactivetosub-strateslackingasidechain[25].ThesubstratespecificityofcholesteroloxidasefromStrep-tomyces cinnamomeuswasstudied[70]inorientedsterolmonolayersanditwasfoundthatthecholesterolanalogue5α-cholestan-3β-olwasoxidizedalmostasfastascholesterolitself.Inaddition,whentheΔ-5doublebondincholesterolwasinsteadattheΔ-4positionandΔ-5doublebondwasattheΔ-7position(5α-cholest-7-en-3β-ol),theoxidationratebecameslower.WithC-17sidechainanaloguesofcholesterol,itwasobservedthatthecompletelackoftheC-17sidechain(5-androsten-3β-ol)ortheinsertionofanunsaturationatΔ-24(desmosterol)orevenanethylgroupatC-24hadnoappreciableeffectsonsteroloxidationrate,implyingthattheenzymedidnotrecognizethesidechaininorientedsterolmonolayers[70].SubstratespecificityofacholesteroloxidasefromRhodococcussp.GK1wasexaminedandtheenzymewas found to bemost activewith cholesterol (100%), β-sitosterol (70%) and stigmasterol(40%).SterolswithmodifiedA-ringsandB-ringsorthe3α-OHofcholicacidremainedun-oxidised[38].CholesteroloxidasefromBrevibacterium sterolicumrapidlyoxidizedcholes-terol,pregnenoloneandβ-sitosterolbutwaslessreactivetowardsstigmasterol.However,itwasfoundtobeunreactivetowardcholicacid,deoxycholicacid,5α-androstan-3α,17β-diolandandrosterone[44].Exceptcholesterol,β-cholestanolwasalsooxidizedatahighratebymostenzymes[23].Thedoublebondinthesteroidringbetween5and6positionsdoesnotseemtobeessentialforthisenzymeactivityandthesterolswiththeshortsidechainareoxi-dizedatalowratecomparatively.Althoughtheoxidationratesofpregnenolonebymosten-zymeswereslow,theenzymesfromChromobacteriumsp.DS-1,Streptomycessp.SA-COOand S. violascensoxidizedpregnenoloneatahighrate[17,71].Chromobacteriumsp.DS-1cholesteroloxidaseoxidizedmost3β-hydroxysteroids.Interestingly,thecholesterolwasthebestsubstrateandβ-cholestanol,pregnenoloneandβ-sitosterolwererapidlyoxidized.Incon-trast,theβ-stigmasterol,ergosterol,dehydroepiandrosteroneandepiandrosteronewereslowlyoxidized.Epicholesterolwascompletelyresistanttoenzymaticoxidation[17].

Cholesterolisacommonlyfoundsteroidwithagreatimportanceinbiology,medicineandchemistryasitplaysanessentialroleasastructuralcomponentofanimalcellmembranes.Cholesterolisfoundnormallyinnaturebecauseofitshighresistancetomicrobialdegradation.


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Owingtoitscomplexspatialconformationandlowsolubilityinwater,cholesterolisaveryresistantmoleculetobiodegradation.Thehighhydrophobicityandlowvolatilityofcholes-terolleadtoahighabsorptiontosolidphases.Becauseofhighrateofpersistence,cholesterolandsomederivedcompoundssuchascoprostanolhavebeenusedasreferencebiomarkersforenvironmentalpollutionanalysis[72].Steroids,someofthemderivedfromcholesterolcon-stituteanewclassofpollutantsdischargedintotheenvironmentasaresultofhumanactivity[73].Thepotentmetabolic activitiesof these compounds affect a largenumberof cellularprocessesandthus,theirpresenceandaccumulationinwater-wastesandincertainecologicalnichescanaffecttheendocrinesystemofanimalsandhumans[74].Moreover,cholesterolisthemaincomponentoflanolinandthisandotherrelatedsterolsarenaturalcontaminantsfairlyresistanttotheanaerobictreatmentthatiscarriedoutontheeffluentsfromwool-processingindustry[75].Ineukaryotes,steroidoxidationandisomerizationareimportantstep(s)inthesynthesisofawidevarietyofsteroidhormonesthatarecarriedoutbyNAD+dependent3β-hy-droxysteroiddehydrogenaseasmembrane-boundproteinlocatedintheendoplasmicreticulumandmitochondrion[76,77].Henceflavin-mediatedcholesteroloxidationisaprocessuniquetomicroorganisms.Itisgenerallyassumedthatthefirstreactionintheaerobicmetabolismofcholesterolisitsoxidationtocholestenonethroughtwosequentialreactions.Thatis,theoxi-dationofcholesteroltocholest-5-en-3-onefollowedbyitsisomerizationtocholestenonebothcatalysedbycholesteroloxidase.

Figure 2:Broaderapplicationsofcholesteroloxidase.


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5. Major Applications of Cholesterol Oxidase

Themicrobialoxidaseshavebeenreportedfrommanybacterialspeciesanddiversehab-itats.Invariably,cholesteroloxidaseshavebeenexploitedinbiotransformation’sofsteroids,biosensors,membranestructuralstudies,insectcontrol&larvicidaldrugsandantimicrobialdrugs(Figure 2).Thebroaderknownapplicationsofcholesteroloxidasesareasfollows;

5.1. Cholesterol biotransformation

Biotransformation’sarestructuralalterationsinachemicalcompoundwhicharecata-lyzedbymicroorganisms in termsofgrowingor restingcellsorby isolatedenzymes.Be-cause of the high stereo- or regio-selectivity combinedwith high product purity and highenantiomericexcesses,biotransformation’scanbetechnicallysuperiortotraditionalchemicalsynthesis.Ifthesefeaturescanbecombinedwitheconomicbenefits,biotransformation’sbe-comethefunctionalpartofnewchemicalprocessesfororganicsynthesis.Furtheradvantagesofbiocatalyticprocessesarethemildandecologicallyharmlessreactionconditions(normalpressure,lowtemperatureandneutralpH),whichareimportantrequirementsforsustainabil-ity.Bioconversionsofhydrophobiccompoundsoftenmeetwithtwoseriousobstacles:limitedsubstrateaccessibilitytothebiocatalystasaresultofthelowaqueoussolubilityofmostorgan-ics;inhibitionortoxicityofbothsubstrateandproductexerteduponthemicroorganism[78].Cholesterolismetabolizedbyalargenumberofmicroorganismsthroughacomplexmetabolicpathwayinvolvingmanyenzymaticsteps,firststepinvolvingtheoxidationofthe3β-hydroxylgroupfollowedbytheoxidationofthe17-alkylsidechainandthesteroidringsystem,finallydegradingtheentiremoleculetoCO2andH2O[23].Inthesequenceofthecholesteroloxida-tionbymicrobialcells,4-cholesten-3-onemaybeoxidizedwithaccumulationofthesteroidsADandADD,whichareimportantprecursorsofchemicallysynthesizedhormonesormaybetransformedtosteroidintermediates[79]. R. equiDSM89-133wasemployedfortheconver-sionofcholesterolandothersterolstoandrost-4-ene-3,17-dioneandandrosta-1,4-diene-3,17-dioneandupto82%ofcholesterolwasconvertedwhenthegrowthmediumwassupplementedwithacetate[80].

BioconversionofcholesteroltoADDandADincloudpointsystemwasstudied[81].CholesterolwasinitiallyconvertedtocholestenonebyArthrobacter simplexU-S-A-18.Cho-lestenonewasprepareddirectlyfromthefermentationbrothofA. simplexandconvertedtoADDbyMycobacteriumsp.NRRLB-3683.ConversionofADtoADDhasbeenreportedinmixedcultureofMycobacterium-Nocardioides[82].Biotransformationofsterolsisinitiatedbymodificationofthe3β-ol-5-ene-to3-oxo-4-enemoiety.Theroleofcholesteroloxidaseinthisprocesshasbeenelucidated.Thecholesteroloxidaseisnotcriticalforsterolcatabolisminthefast-growingAD-producingMycobacterium sp.VKMAc-1815Dstrainandtheknock-outofcholesteroloxidasegenedidnotabrogatesterolring-Aoxidation[83].Similarconclusions


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weremadeearlierfor Mycobacterium smegmatismc2155[84].In Rhodococcus erythropolis CECT3014,cholesteroloxidasewasshowntobeamajorinducibleextracellularcholesteroloxidasebutitsdisruptiondidnotaltercellgrowthoncholesterol[85].However,inStrepto-myces virginiaeIBL-14,inactivationofcholesteroloxidaseledtoabrogatetheoxidationofdiosgenintodiosgenoneandother3-oxosteroids[86].Targetedgenedisruptionofcholesteroloxidases in Gordonia cholesterolivoransCECT7408Tresultedinamutantstrainunabletogrowonsteroids.Twocholesteroloxidases,ChoM1andChoM2wereidentifiedinMycobac-terium neoaurumNwIBandtheseweresuggestedtobeessentialforutilizationofphytosterolasacarbonsource[87].Alongwithcholesteroloxidases,3β-hydroxysteroiddehydrogenases(3β-HSDs)cancatalyse3β-hydroxygroupoxidationandΔ5→4isomerizationinactinobacte-ria[84].

Mycobacterium smegmatisPTCC1307wasusedasamicrobialagenttoproduceADDandAD,twousefulprecursorsinthesynthesisofsteroiddrugs.Thesidechainofcholesterol,asthesubstrate,wasselectivelycleavedinthepresenceoffiveenzymeinhibitors.Aninterme-diatestructurewithintactsidechain,cholest-4-ene-3-one,wasalsodetectedandpurified[88].Wuandcoworkers(2015)exploredproductionofcholest-4-en-3-onebydirectlyusingcholes-teroloxidasefromRhodococcussp.inanaqueous/organictwo-phasesystemanditwasfoundthattheconversionwasmoreefficientinthebiphasicsystemthanintheaqueousorco-solventsystem.After48hofreaction,theconversionratereached94.2%inthebiphasicsystemandonly42.3%conversionwasachievedintheaqueoussystem[14].Inanotherstudybiotransfor-mationofcholesteroltoADDbycholesteroloxidasefromChryseobacterium gleum was stud-iedandthegrowingcellsproduced0.076gADDfrom1gcholesterol,whichwasequivalentto10%molarconversionofcholesterol[89].Whole-cellsoftherecombinantstrainsBacillus subtilis168/pMA5-choM1andB. subtilis168/pMA5-choM2expressingchoMgeneencodingcholesteroloxidasefromMycobacterium neoaurumJC-12wereusedascatalystsforthebio-conversionofcholesterolto4-cholesten-3-oneandapercentageconversionof67%and83%wasobservedat21h[90].R. erythropolischolesteroloxidasewasemployedfortheprepara-tiveoxidationofcyclicallylic,bicyclicandtricyclicalcoholsandforthesynthesisofseveralergotalkaloids[63].

5.2. Therapeutic uses

Cholesteroloxidaseisusedasanefficientanalytictoolfordeterminingcholesterolinvarioussamples;totalandesterifiedcholesterolinserumorblood,fromlow-densitylipopro-teinstohigh-densitylipoproteins,onthecellmembrane,ingallstones,inhumanbileandinvariousfoodsamples[21].Normalcholesterollevelinhumanbloodislessthan200mg/dLandlipoproteinscontaincholesterolofwhich~70%isinesterifiedform.Highlevelsofcholes-terolandcholesterylesters(hypercholesterolemia)havebeenassociatedwithcardiovasculardiseasesuchasatherosclerosisandotherheartdiseases,althoughlowerlevels(hypocholester-


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olemia)maybeassociatedwithcancer,depressionorrespiratorydiseasesmakingdeterminingofserumcholesterolconcentrationveryimportant.Alzheimerdiseaseriskisalsoreportedtoberelatedtohypercholesterolemiavia involvingoxidativestressmechanisms[91].Choles-terollevelinserumisgenerallydeterminedbyusinganenzymaticassay[48,69].Asmostofcholesterolinserumsamplesexistsinanesterifiedformsopriorincubationofserumwithcho-lesterolesterase(EC3.1.1.13)isrequiredtoreleasethefreecholesterol.CholesteroloxidasecatalyzesoxidationofcholesteroltocholestenonewithsimultaneousreleaseofH2O2.Theen-zymesubsequentlycatalyzestheoxidativecouplingofH2O2withachromogenicdyewhichisdeterminedspectrophotometrically.Inadditiontobeingusedinthemicroanalysisofsteroidsinfoodsamplesit isalsousedfordifferentiatingthestericconfigurationsof3-ketosteroidsfromthecorresponding3β-hydroxysteroids[92].Overtheyears,variouselectrochemicalbio-sensorshavebeendesignedbyusingimmobilizedcholesteroloxidaseforthedeterminationofcholesterolinserumorfoodsamples[93-95].

5.3. Insecticidal activity

Geneticallymodifiedplantswhichareabletocontrolinsectpestsbyproducinginsecti-cidalproteins(suchasBacillus thuringiensistoxin)arebeingusedverywidelytoreplacetheuseofsyntheticpesticides.Ahighlyefficientbacterialproteincapabletokill larvaeofbollweevil(Anthonomus grandis grandisBoheman)intheculturefiltratesofStreptomyces sp.wasidentifiedasacholesteroloxidase[15].Whenthisenzymewasusedinpurifiedformagainstbollweevillarvae,itshowedgoodactivityatlethalconcentrationof50%(LC50of20.9μg.ml),which iswellcomparable to the insecticidalbioactivityexhibitedbyBacillus thuringiensis proteins.Cholesteroloxidaseinsecticidalactivityisreportedduetoinductionoflysisatthemidgutepitheliumoflarvaeuponingestion.Cholesterolorsomeotherrelatedsterolatthebollweevilmidgut epitheliummembrane appeared tobe available for oxidationby cholesteroloxidase,causinglysisofthemidgutepithelialcellsresultinginlarvaldeath[23].Theenzymealsoshowedinsecticidalactivityagainstlepidopterancottoninsectpests, includingtobaccobudworm(Heliothis virescens),cornearworm(Helicoverpa zea)andpinkbollworm(Pectino-phora gossypiella)[96].Microbialinsecticideproteinsareveryimportantinseveralpestcon-trolstrategiesemployedintransgeniccropsandcholesteroloxidasegenefromStreptomyces sp.hasbeenexpressedintobaccoprotoplasts[97].

5.4. Cell membrane structural studies

Cholesterol is themain sterol constituent of eukaryotic cellmembrane essential formaintainingcellmembranestructureandstability.Thecellsurfacecholesterolcanbedeter-minedaccuratelywithcholesteroloxidasehencethesurfaceareaofagivencellcanbeesti-matedreadily[98].Cholesteroloxidaseisusedasaprobetoinvestigatecholesterolinteractionwithphospholipidsascholesterolassociatespreferentiallywithsphingolipidsincholesterol-


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richlipidraftsareasofthemembranesineukaryoticcells[100,101].Thelipidraftsarethemi-crodomainsinwhichcholesterolandsaturatedmembranelipidssuchassphingolipidspromotetheformationofahighlyorderedmembranestructureincomparisontothemoredisorderedvicinity[102,103].Lipidraftsplayrolesinvariouscellularprocesses,includingsignaltrans-duction,proteinandlipidsorting,cellularentrybytoxinsandviruses,andviralbudding[23].Therefore,cholesteroloxidaseplaysasignificantroleinthestudyofthefunctionalaspectsoflipidraftwithregardtoeukaryoticmembrane.

5.5. Potential new antimicrobial drug target

Awidevarietyofmicroorganismsincludingsomelifethreateningpathogenicbacteriasuch as Rhodococcus equi, Mycobacterium tuberculosis and M. lepraearereportedtoproducecholesteroloxidase[104].Rhodococcus equiisaGram-positivebacteriumknownforcausinginfectioninyounghorsesaswellasinhumansactingasanopportunisticpathogeninimmuno-compromisedpatients[36,105].Cholesteroloxidasemaypossiblyactsasaninterestingphar-maceuticaltargetfortreatingbacterialinfections.In vitrostudiesadvocatedthatduringR. equi infectionofthehostcell,membranelysisisfacilitatedbytheinductionofextracellularcho-lesteroloxidasealongwithothercandidatevirulencefactors[35].Themembrane-damagingactivityofR. equirequiresthepresenceofbacterialsphingomyelinaseCwhichsuggestedthatthecholesteroloxidasesubstrateisnotdirectlyaccessibletotheenzymeinintactmembranes[21].IthasalsobeenreportedthatcholesteroloxidaseisalsoinvolvedinthemanifestationofHIVandnonviralprionorigin(Alzheimer’s)diseases[106].Thepathogenicbacteriautilizecholesterol oxidase for infection by converting the cholesterol ofmembranes thus causingdamagebyalteringthephysicalstructureofthemembrane.Theemergingproblemofanti-biotic resistantbacteriaand theirabilities for rapidevolutionhavepushed theneed tofindalternativeantibioticswhichare lessprone todrugresistance.Sincenoeukaryoticenzymehomologuesexist, thistypeofbacterialcholesteroloxidasefallsintothescopeofpotentialdrugtargetforanewclassofantibioticswhichstillremaintobeexplored[107].

5.6. Polyene macrolide pimaricin biosynthesis

TheStreptomyces natalensischolesteroloxidasewhichisaproductofthepimEgeneplaysanimportantroleinthebiosynthesisofthepolyenemacrolidepimaricinbyactingasasignalingmolecule[108].This26-membertetraenemacrolideantifungalantibioticisusedinthefoodindustrytopreventcontaminationofcheeseandothernon-sterilefoodwithmoldandalsofortreatingthefungalkeratitisbecauseofitsabilitytointeractswithmembraneste-rolsespeciallyergosterol,resultinginthemembranestructurealterationthatcausesleakageofcellularcontents[21].Thepolyenemacrolidepimaricingeneislocatedinthecenterofthepimaricinbiosyntheticclusterasthepimaricinproductioniscompletelyblockedbythegenedisruptionwhichisrecoveredaftergenecomplementation.TheadditionofpurifiedPimE or


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exogenouscholesteroloxidasetothegenedisruptingculturecanrestorethepimaricinproduc-tion.Thesefindingssuggestedtheinvolvementofcholesteroloxidasesassignalingproteinsforpolyenebiosynthesis[23].

5.7. Cholesterol oxidase as a novel antitumor therapeutic drug

Cholesteroloxidasecatalyzesoxidationofcholesterolandhasbeenusedtotrackmem-branecholesterol.Liuandcoworkers(2014)reportedthatcholesteroloxidasefromBordetella species madelungcancercellsbothin vitro and in vivotoundergoirreversibleapoptosis.Cho-lesteroloxidase treatment inhibitedphosphorylationofAkt(proteinkinaseB)andERK1/2(extracellularsignal-regulatedkinase1/2)whichwasirreversibleevenaftercholesteroladdi-tion.Furtherstudiesindicatedthatcholesteroloxidasetreatmentalsopromotedthegenerationofreactiveoxygenspecies(ROS).InadditiontothischolesteroloxidasetreatmentresultedinphosphorylationofJNK(c-JunNH2-terminalkinase)andp38,downregulationofBcl-2(B-celllymphoma/leukemia-2),upregulationofBaxwiththereleaseofactivatedcaspase-3andcytochromeClikelyduetoproductionofhydrogenperoxidealongwithcholesteroloxidation.Thesefindingssuggestedthatcholesteroloxidaseleadstoirreversiblecellapoptosisbyde-creasingcholesterolcontentandincreasingROSlevelindicatingcholesteroloxidasemaybeapromisingcandidateforanovelantitumortherapy[109].

5.8. Probiotics

Probiotics are livingmicroorganismswhichupon ingestion in certainnumbers exerthealthbenefitsonthehostbeyondinherentbasicnutrition[110].Thoughseveralin vitro and in vivostudieshaveprovedthattheadministrationofprobioticsdecreasesserum/plasmatotalcholesterol,LDL-cholesterolandtriglyceridesorincreasesHDL-cholesterolbuttheirhypoc-holesterolemiceffectsstillremaincontroversial[111].Thespecieswhichhavebeenfoundtoexertcholesterol-loweringeffectsincludegeneralikeLactobacillus, Lactococcus, Enterococ-cus, Streptococcus and Bifidobacterium[112].Differentmechanismslikeco-precipitationofcholesterolwithdeconjugatedbile, cholesterolbinding to cellwalls, integrationof choles-terol into thecellularmembranesduringgrowth,productionof short-chain fattyacidsdur-ingfermentationandtransformationintocoprostanolareproposedforcholesterol loweringeffectsofprobiotics [112-115].Cholesteroloxidase,especiallyderived frombacterialcellsplaysamajorroleinthedegradationofcholesterolinmanyfermentedfoods[116].Khiralla,(2015)reportedthatLeuconostoc mesenteroidesGMK03coulddegradecholesterolusingtheextracellularcholesteroloxidaseandfoundtobetoleranttolowacidity(pH2.5)andbilesalts(0.3%),competenttoadhesiontoCaco-2cellsandhavelowantibioticresistance,socouldbeconsideredasapromisingprobioticstrain[117].Ageneencodingcholesteroloxidase(choA)fromStreptomyces[118]hasbeenexpressedinseveralspeciesofprobioticbacteriaincludingLactobacillus casei[119],Lactobacillus reuteri[120]andStreptococcus thermophilus[121].


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Rossietal.,(1998)reportedthecloningofcholesteroloxidasefromStreptomyces sp.intoBifi-dobacteriumunderitsownpromoterbutwithoutsuccessfulexpression[122].Parketal.,suc-cessfullyclonedandexpressedcholesteroloxidasegenefromaStreptomyces sp.inpromoterandRBS(ribosome-bindingsite)ofBifidobacterium longumMG1[123].Bacterialdegrada-tionofcholesterolincholesterolcontainingfoodmaybebeneficialforhumanhealth[124].

5.9. Cholesterol oxidase biosensors for analytical assays

Cholesterolisanimportantanalytemoleculeanditsassayisimportantforthediagnosisandpreventionofanumberofclinicaldisordersandinfoodanalyses.Moreover,quantifica-tionofcholesterolpresentinvariousfoodsisvitalforselectingadietwithoptimalintakeofcholesterol.Thus,itisnecessarytodevelopnewtechniquesforeasyandrapidestimationofcholesterollevelsinvariousanalyticalsamples[125].Biosensorswithimmobilizedenzymesareofhugeinterest inthevariousanalyticalproceduresastheyfacilitatetheenzymereusealongwithexclusiveselectivityofthebiologicalmoleculesandtheprocessingpowerofmod-ernmicroelectronics [126].Overaperiodof time,avarietyofcholesterolbiosensorshavebeen developed [95].Most of the reported enzyme-based cholesterol biosensors are fabri-catedonamperometrictechniquebesides,cholesterolbiosensorsbasedonphotometricbehav-iorslikeluminescence,fluorescenceandsurfaceplasmonresonancehavealsobeenreported[95,127,128].AmperometricmeasurementofO2consumptionorH2O2productionduringcho-lesterolcatalysisbycholesteroloxidaseisthefrequentlyusedstrategyincholesterolbiosensors[129].Acholesterolbiosensorbasedoncholesteroloxidase-poly(diallyldimethylammoniumchloride)-carbonnanotubes-nickelferritenanoparticles(ChOx-PDDACNTs-NiFe2O4NPs)wasfabricatedbyusingasingledroppingsteponaglassycarbonelectrodesurface[130].Variousnanoparticleslikemetalnanoparticles(gold(Au),platinum(Pt),silver(Ag)NPs),metalox-idenanoparticles(zincoxide(ZnO),ironoxide(Fe3O4),ceriumoxide(CeO2),titaniumoxide(TiO2)NPs),carbonnanotubes(single-walledcarbonnanotubes(SWCNTs),andmulti-walledcarbonnanotubes(MWCNTs)basedmaterialshavealsobeenexploitedfordesigningcholes-terolbiosensor[131-134].

6. Conclusion

Cholesteroloxidasehasbeenreportedfromalargenumberofbacterialspeciesandtheactinomycetesbeingthemostcommongroup.Beinganenzymeofgreatcommercialvalue,cholesteroloxidasehasdrawnsignificantattentionduetoitswidespreaduseindeterminationofcholesterollevel(s)invariousclinicalandfoodsamplesandbecauseofitsothernovelap-plicationslikebiocontrolofinsects,polyenemacrolidepimaricinsynthesis,biocatalysisforthesynthesisofanumberofsteroidsandfabricationofbiosensors.Biochemicalandstructuralstudiesoncholesteroloxidasecanplay important roles inunderstandingofvariouscataly-sisaspectsofdifferentflavoenzymesasitbelongstoflavoproteinoxidases.Thuscholesterol


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oxidaseisaversatileenzyme,thenewermicrobialsourcesinnatureneedtobeexploredforsynthesisofextracellularcholesteroloxidase(s),whichis/areeasytopurify,characterizeandputforextendedcommercialuses.

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Current Research in Microbiology - Open Access. eBooks · Current Research in Microbiology 5 to be optimally active at neutral pH and are stable over a wide pH range. The enzymes