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REVIEWOpenAccess Enzymepromiscuity:usingthedarksideofenzyme specificityinwhitebiotechnology BenuArora 1 ,JoyeetaMukherjee 1 andMunishwarNathGupta 2* Abstract Enzymepromiscuityresultsinfarlargerrangesoforganiccompoundswhichcanbeobtainedbybiocatalysis.While earlyexamplesmostlyinvolveduseoflipases,morerecentliteratureshowsthatcatalyticpromiscuityoccursmore widelyandmanyotherclassesofenzymescanbeusedtoobtaindiversekindsofmolecules.Thisisofimmense relevanceinthecontextofwhitebiotechnologyasenzymecatalysedreactionsusegreenerconditions. Keywords: Enzymespecificity,Catalyticpromiscuity,Enzymesinorganicsynthesis,Enantioselectivity,Greenchemistry “ IsuspecttheyputSocratestodeathbecausethereis somethingterriblyunattractive,alienatingandnon- humaninthinkingwithtoomuchclarity. ” - TheBedofProcrustesbyNassimNicholasTaleb Introduction Thebasictenetofwhitebiotechnologyistominimize damagetotheenvironmentratherthantakingrecourse toremediationasan “ endofthepipe ” solution[1,2].En- zymeseitherinisolatedformorintheformofwholecells canplayanimportantrolebecauseoftheirtwowell knownvirtues.Thebiocatalystsnormallydonotrequire hightemperatureorotherconditionswhichinvolvehigh consumptionofenergy.Thebiocatalystsarebelievedto befairlyspecific,whichwouldmeanlessnumberofside theatomeconomyofthereactions.Thesesidereactions hencelowertheyieldofthedesiredproduct(makingthe catalysislessefficient)andnecessitatecomplicateddown- streamprocessing. Thisreviewdiscussestheparadigmshiftsovertheyears inourunderstandingoftheenzymespecificity.Italsoex- plainswhysocalledlackofspecificityisalsoagoodnews asfarastheusefulnessofenzymesinbiotechnology isconcerned.Themostdramaticdeparturefromthe classicalconceptofenzymespecificityisseeninthe phenomenonofcatalyticpr omiscuity.Thisrefersto thesameenzymecatalyzingverydifferentkindsofbio- transformations[3-6]. Classificationofenzymesandenzymespecificity Mostofthepeoplehaveforgotten(understandablesinceit disappearedfromthestandardtextsmanydecadesback!) thatinitiallyproteinswereclassifiedaccordingtotheir solubilityinvarioussolvents.Thefiveclassesofproteins werealbumins(solubleinwaterandsaltsolutions),globu- lins(sparinglysolubleinwaterbutsolubleinsaltsolu- (solubleinacidoralkali)andscleroproteins(insolublein aqueoussolvents)[7].Asourknowledgeofproteinsgrew, thedistinctionbetweenthesevariousclassesbecamefluid andinfactconfusing.Theenzymes,intheearlyphase ofbiochemistrybecamethemostimportantandmost studiedclassofproteins.Thesewerenamedinarandom fashionjustaspeoplenamebuildings,streetsandmonu- ments.Manyofthesenamespersiste.g.catalase,trypsin, lysozymeetc.Manyofthesenameswerebaseduponthe natureoftheircatalyticactivity.Lysozymeisnamedsoas itlysescells.So,thenomenclatureandcatalyticspecificity havealonghistoryintheareaofbiocatalysis. Theideaofaparticularstructurebeingabsolutelyrelated toabiologicalactivityhasbeenconsideredthehallmarkof biologyformanydecades.Hence,itisnotsurprisingthat enzymecommissionclassificationwasbaseduponthetype *Correspondence: munishwar48@yahoo.co.uk 2 DepartmentofBiochemicalEngineeringandBiotechnology,IndianInstitute Fulllistofauthorinformationisavailableattheendofthearticle ©2014Aroraetal.;licenseeSpringer.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle, unlessotherwisestated. Arora etal.SustainableChemicalProcesses (2014) 2:25 DOI10.1186/s40508-014-0025-y ofreactiontheenzymecatalyzes – hydrolysis,isomerisation orredoxreactionsetc.[8].Thisinretrospectmaynothave beensuchawisemoveaswebelievedtillnow. In1955 – 56,InternationalCommissionofenzymeswas setupandthatishowtheECclassificationcameintobe- ing[8].Asweknow,thisclassificationprovidesanumber withfourfigures.Itisworthnotingthatallthefourfigures relatetothedetailsofthespecificityoftheenzyme.The firmbeliefwasthatifoneisabletopindowncomplete detailsofthespecificity,onehasdescribedtheenzyme andthatisitsidentity. Variouskindsofenzymepromiscuity Nothingpromotesascientificdirectionasmuchascoining anewterm.Systembiology,nanotechnologyandsynthetic biologyareillustrativeexamples.Enzymepromiscuityis alsoacaseinpoint.AsHultandBurglund[9]pointed out,promiscuouscatalysisbypyruvatedecarboxylaseof formationofC-Cbondswasreportedin1921andforms thebasisofacurrentindustrialprocess.Khersonskyand Tawfik[4]havealsocitedexamplesoffewotherwell knownenzymeswhich,manydecadesback,werereported tocatalysereactions “ inadditiontotheonesforwhich theyarephysiologicallyspecializedorevolve …” .AsBabtie etal.[10]highlighted “ Promiscuousactivitiesaregenerally considerablylessefficientthantheprimaryfunctionofan enzyme,butsecondorderrateconstants(k cat /K M )ashigh as10 5 M  1 s  1 andrateaccelerations((k cat /K M )/k 2 )upto 10 18 havebeenreported:thesevaluesmatchorexceed thoseformanynativeenzymaticreactions. ” .Afterinitial looseusagesoftheterms(associatedwithpromiscuous behaviourofenzymes),thereisclaritythatbroadspecifi- cityofanenzymeintermsofcatalysisofthesamereac- tionwithrangeofsubstratesshouldbecalledsubstrate promiscuity.Instanceswhenanenzymecatalysesadiffer- entreaction(whichisnotbelievedtobephysiologically relevantatthatpointinevolution)withadifferenttransi- tionstateshouldbetermedasexamplesofcatalyticprom- iscuity[9,10]. HultandBerglund[9]goastepfurtherandsuggestthat reversereactionscatalysedbyenzymesinmanynon- aqueousmediaorco-solventscanbeclassifiedascondition promiscuity.Insuchcases,thetransitionstatemaybesame asinnormalreactions.So,thismaybelittleconfusing. Enzymepromiscuitystartedasbeingperceivedas “ asso- ciatedwithunwantedsideeffects,poorcatalyticproperties anderrorsinbiologicalfunction ” [11].Today,itisincreas- inglybeingperceivedasimmenselyusefulphenomenon whichcandramaticallyenhanceutilityofbiocatalysisin biotechnology. Babtieetal.[10]haveprovidedagooddiscussionon howcatalyticpromiscuitymayoperate.Apartfromthe differentaminoacidsoftheactivesiteinvolvedina qualitativeorquantitativefashionduringbindingofthe “ alternativesubstrates ” ,hydrophobicinteractionsmay haveanimportantroletoplay.Notonlyduringbinding, evenduringcatalyticsteps,thecontributionsoftheac- tivesiteresiduesmaydifferqualitativelyand/orquanti- tatively.This “ accidentalpromiscuity ” observedinwild formsoftheenzymesshouldbedistinguishedfrom “ in- ducedpromiscuity ” exhibitedbymutantsobtainedby proteinengineeringordirectedevolution[12].Manyex- cellentreviews(otherthanthosealreadyquotedabove) whichincludeevolutionaryaspectsofpromiscuityare available[3,13,14]. Conditionpromiscuityofenzymes Hydrolaseshydrolysesubstrates.Whathappensifwater isnearlyabsent?Adventofnon-aqueousenzymology showedthatexploringthispossibilityledtocarryingout biocatalysisinnearlyanhydrousorganicsolvents[15,16], reversemicelles[17,18],ionicliquids[19-22]etc.So,a lipaseinlowwatercontainingorganicsolventsynthe- sizesanesterbondandisobviouslynotfunctioningasa hydrolase[23]. Thishasbeenaveryusefuldiscovery.Ithasbeenalso veryimportantasitopeneduphugepossibilitiesof usingenzymesinbiotransformations[16,24][Figure1]. Notonlyitmakesitpossibletouseinexpensivehydrolases likeproteasesandlipasesinorganicsynthesis,itprovides anadditionalhandleofmediumengineering[25,26].Chan- gingtheorganicsolventmaychangetheenantioselectivity [27,28].Theotherpossibilitiesemergede.g.usingorganic solventsasco-solvents[29]orusingbiphasicsystemscon- sistingofaqueousmediumandwaterimmiscibleorganic solvents[30].Onecanuseorganicsolventphasetodis- solvesubstratesandaqueousphasecancontaintheen- zyme.Thismakesthecatalyticprocessveryefficientin casethesubstrateinhibitionisinvolved[31].Iftheprod- uctalsogoesbacktotheorganicphase,shiftingofthe equilibrium(infavourofproductformation)andover- comingproductinhibition(ifinvolved)arebothachieved. Morenewpossibilitiescontinuetoemerge.AsDordick ’ s groupshowedfewyearsback,nanotubemediatedassem- blyofenzymesattheinterfaceofaqueousandorganic mediaovercomesthetransportlimitationstypicalofsuch biphasicsystems[32]. Itincidentallyalsomeantthatonecouldusegreensol- ventsforbiocatalysissuchasglycerol,ethanol,succinate esters,lactateesters,limoneneandsupercriticalfluids [34].Thecaseofglycerolisespeciallyrelevantinthecon- textofwhitebiotechnologyasmassiveamountsofgly- cerolaregeneratedduringbiodieselproductionandhence fitsinwellwiththebiorefineryconcept.Thebiodieselas suchcanbeobtainedfromwastesandcanbeagoodex- ampleofwastevalorization[35-37][Figure2].Manyof theabovementionedsolventsalso,inprinciple,canbeob- tainedfromnonfoodbiomassorwastefoodmaterials Arora etal.SustainableChemicalProcesses (2014) 2:25 Page2of9 [35].Allthispointsouttotheemergingcontoursofa chemicalindustrybaseduponsustainablepractices. Thesepossibilitieshaveemergedasourclassicalcon- ceptofhydrolaseswillbealwayshydrolasesturnedout tobenotabsolutelyvalid.Ifyouchangethereaction condition,enzymesshowconditionpromiscuity.That hasnotturnedouttobebadatallfromtheperspective ofwhitebiotechnology. Catalyticpromiscuity:recentresults Asmanyexamplesofcatalyticpromiscuityhavebeencov- eredinquiteafewrecentreviews[5,38,39],wewillmostly focusonliteraturepertainingtolastcoupleofyears.Rather thanaimingatbeingcomprehensive,thethrustofthis reviewisonillustratinghowcatalyticpromiscuitycanbe exploitedtocreatenovelpossibilitiesintheareaofbioca- talysisdrivingthegrowthofwhitebiotechnology.Someex- amplesofreactionscarriedoutusingcatalyticpromiscuity forthesynthesisoforganiccompoundsintheauthors ’ la- boratoryareillustratedinTable1.Also,wehavemostly limitedourselvestoexamplesofaccidentalpromiscuityra- therthanincludingevergrowingnumberofexamples wherecatalyticpromiscuityhasbeentailoredbyusingpro- teinengineeringordirectedevolution. Catalyticpromiscuityoflipases Singlepotmulticomponentreactionsareanefficientsyn- theticstrategyespeciallyifaccompaniedbygoodatom Figure1 Lipasecatalyzedtransesterificationof
,  -glucoseor
,  -maltosewithvinylpropionateorethylacrylateasacyldonorproduced monoestersanddiesters.
-Cyclodextrinandthementionedesterproducts,weresubstratesinthesecondreactioncatalyzedbyCGTase from B.macerans toproduceoligosaccharideesters.[Reproducedfro mRef[33]withpermissionfromChemistryCentral]. Figure2 BiodieselconversionwithcleanoilusingNovozym435+RMIM. Reactionswereperformedtaking0.5gcoffeeoil.Ethanolwas addedinmolarratio4:1(ethanol:oil).Novozym435+RMIM=3:1(WhiteBars);37.5mgofenzymeNovozym435(enzymeloadbeing7.5%w/w ofoil)and12.5mgofenzymeRMIM(enzymeloadbeing2.5%w/wofoil)wasadded.Novozym435+RMIM=1:1(GreyBars);25mgofenzyme Novozym435(enzymeloadbeing5%w/wofoil)and25mgofenzymeRMIM(enzymeloadbeing5%w/wofoil)wasadded.Novozym435+ RMIM=1:3(Blackbars);12.5mgofenzymeNovozym435(enzymeloadbeing2.5%w/wofoil)and37.5mgofenzymeRMIM(enzymeload being7.5%w/wofoil)wasadded.[ReproducedfromRef[37]withpermissionfromChemistryCentral]. Arora etal.SustainableChemicalProcesses (2014) 2:25 Page3of9 Table1Someinterestingexamplesoforganiccompoundssynthesizedintheauthors ’ laboratoryexploitingcatalytic promiscuity StructureofthecompoundSubstrateandreactionconditionsReference Substrate: 5-Hydroxy-endotricyclo[5.2.1.0 2,6 ]deca-4,8-dien-3-oneandvinylacetate[ 40 ] Biocatalyst: Novozyme435 Solvent: Vinylacetate(substrate)containing10%(v/v)pyridine/DMF Substrate: p -nitrobenzaldehydeandethylacetoacetate[ 41 ] Biocatalyst: Lipasesfrom Candidaantarctica lipaseB ,Mucorjavanicus,Mucormiehei, Candidarugosa andNovozyme435 Solvent: Aqueousorganicco-solventmixtureswereusedasthesolvent Substrate: p -nitrobenzaldehydeandacetylacetone[ 42 ] Biocatalyst: Lipasesfrom Candidaantarctica lipaseB ,Mucorjavanicus,Mucormiehei, Burkholderiacepacia andNovozyme435 Solvent: Aqueousorganicco-solventmixtures Substrate: p -nitrobenzaldehyde[ 43 ] Biocatalyst: Novozyme435 Solvent: Aqueousbuffer Arora etal.SustainableChemicalProcesses (2014) 2:25 Page4of9 economy.Zhang[44]showedthatporcinepancreatic lipase(PPL)couldbeusedtosynthesizeanumberof 2,4-disubstitutedthiazoleswithmethanolastheorganic solvent.Theirinterestinsynthesisofthesecompoundswas duetotheirmanybiologicalactivities.Thereareseveral noteworthyfeaturesofthiswork.Useoforganicsolvents (ascomparedtowateroraqueous-organicco-solventmix- tures)hasbeenunderexploitedinthecontextofcatalytic promiscuityofenzymes.Moreimportant,someamylase preparationshavealsobeenshowntobecatalysingthisre- action.Itisverylikelythatenzymesusedinthiswork wereindustrialgradepreparationsandmayhavebeen contaminatedbylipaseactivities.Earlier,ittookseveral decadesbeforeNakajima ’ sgroupcouldshowthatlipases (contrarytoanearlierclaim)ifpurifieddonotcatalyse peptidesynthesisinorganicsolvents[45].Asweexplore moreandmorecatalyticpromiscuityofenzymes,itisne- cessarythatwekeepinmindthatsomeofthesemaybe causedbycontaminatingproteins.Anotherimportant pointisthatmethanolasasolventwasfarsuperiorto ethanol.Thatisunfortunateasethanol,unlikemethanol, isagreensolvent.So,fromsustainabilitypointofview, perhapsweneedtoseparatelyscreenoutapanelofgreen solventsanddeterminewhichoneisthebest,evenifnot asgoodasanothernongreensolvent. Ugireactionisoneofthemorewellknownmulticom- ponentreactions[46].TheclassicUgireactioninvolves condensationofaprimaryamine,acarbonylcompound, carboxylicacidandisocyanide.Kzossowskietal.[47]have shownthatNovozym435couldformapeptidebond inorganicsolventsliketolueneandchloroform.An importantobjectiveofwhitebiotechnologyhasbeen tobeabletooperatechemicalprocessesstartingwith simplesetofcompounds[34]whichcanbeobtained fromrenewableresources.Inthatrespect,peptidebond formationstartingwithanamine,aldehydeandiso- cyanide(ratherthantwoaminoacids)isagoodadvance- mentandshowsthewiderangingpromiseandpotential ofcatalyticpromiscuity. Catalyticpromiscuity:beyondlipases Thephenomenonofcatalyticpromiscuityhasturnedout tobefarmorewidespreadthaninitiallybelieved.While earlier,therewasastrongfocusontheuseoflipasesfor C-Cbondformationreactions[38,40,41,48,49],useof otherenzymesincatalyzingdiversekindsofreactionsis beingincreasinglydemonstrated.Liuetal.[50]havere- portedasymmetricaldolreactionsbetweenisatinderiva- tiveswithcyclicketonescatalyzedbynucleasep1from Penicilliumcitrinum .Thisexampleofcatalyticpromis- cuityprovidesagreenapproachforthesynthesisofpharma- ceuticallyactivecompounds.Lietal.[51]hadearlierused thesameenzymeforcatalyzingasymmetricaldolreac- tionsbetweenaromaticaldehydesandcyclicketones undersolvent-freeconditions.Thecatalyticpromiscuity of Escherichiacoli BioHesterasewasrecentlyexploited forthesynthesisof3,4-dihydropyranderivatives[52].The authorsusedaseriesofsubstitutedbenzalacetonesand1, 3-cyclicdiketonesasreactantsinanhydrousDMF,with yieldsashighas76%beingobtainedinsomecases.Re- cently,ficinfromfigtreelatex(aplantcysteineproteinase) wasshowntocatalyzethedirectasymmetricaldolreac- tionsofnitrogen,oxygenorsulphurcontainingheterocyc- licketoneswitharomaticaldehydesinorganicmedium [53].Earlierworkfromthesamegroupshowedthatcom- merciallyavailablepapain(from Caricapapaya latex)can beusedasanefficientcatalystforKnoevenagelreactions involvingawiderangeofsubstrates[54].Zhengetal.[55] developedthetrypsincatalyzedone-potmulticomponent synthesisof4-thiazolidinonesasanovelstrategyforthe synthesisofthisimportantgroupofheterocycliccom- pounds.Thederivativesof2H-1-benzopyran-2-oneform thescaffoldofmanypharmaceuticals[56,57].Wangetal. [58]haveusedanalkalineproteasefrom B.licheniformis toobtainthesebydominoKnoevanagel/intramolecular transesterificationreactionsinlowwatercontainingor- ganicsolvents. Aninterestingexampleofexploitingcatalyticpromiscu- ityforcarryingoutdomino,single-potreactionswasre- portedbyZhouetal.[59].Theauthorsused
-amylase from Bacillussubtilis forcatalyzingtheoxa-Michael/aldol condensationforthesynthesisofsubstitutedchromene derivatives.Gaoetal.[60]describedHenryreactionscata- lyzedbyglucoamylasefrom Aspergillusniger .Thereac- tionscarriedoutinmixedsolventsofethanolandwater, formedthe  -nitroalcoholsinyieldsupto99%. Oneemergingconcernhasbeenthebacteriadevelop- ingresistancetowardsexistingantibioticslikeinthecase ofaminoglycosideswhicharebroadspectrumantibiotics [61].Thecatalyticpromiscuityofaminoglycosideacetyl transesteraseswasutilizedforchemoenzymaticsynthesis ofvarietyofnovelmoleculesincludingacylatedamino- glucosideswhichshowpromiseincircumventingbacterial resistancetowardsexistingantibiotics.Thesyntheticstrat- egyavoidslongermultistepprocesses.Werneburgetal. [62]haveusedthepolyketidesynthetaseforpreparative scalesynthesisof15newaureothin(ashikimate-polyketide withantimicrobialandantitumoractivities)analogs,many withlesscytotoxicitybutimprovedanti-proliferative action.Itmaybenotedthatengineeredmutantsofthe organismwereused.So,itisanexampleofmetabolicen- gineeringwhereinthestrategywasbaseduponthecata- lyticpromiscuityoftheenzyme. Alcoholdehydrogenases(ADHs)areenzymeswhich catalyzethetransformationofketones/aldehydestothe correspondingalcoholsandviceversaattheexpense ofanicotinamidecofactorthatactsashydridedonor andacceptorrespectively[63].Ofthemanyapproaches Arora etal.SustainableChemicalProcesses (2014) 2:25 Page5of9 availableforco-factorrecycling,asimplewayistousea co-substratesuchaspropanol[64,65].Gotor ’ sgroup[66] hasusedADHsfrom L.brevis,R.ruber and Thermoanaer- obacter sp.tocarryoutregioselectiveandstereoselective reductionsof1,2-and1,3-diketonestoobtainenantio- purehydroxyketonesordiols.Somecyclicdiketoneswere alsoreduced.Theco-substratepropanolwasusedforco- factorregenerationinthiscaseaswell.Whilethereaction carriedoutwasessentiallyanormalone,thebinding modesofthesubstratesweredifferentwithdifferent ADHsandshowhowdifferentpartsoftheactivesiteof theenzymescanbeinvolvedinbinding.Thisisagoodil- lustrationofsubstratepromiscuitybeingexploitedforde- signingusefulsyntheticstrategies.Aninterestingexample ofcatalyticchemo-promiscuityofalcoholdehydrogenase wasreportedbyFerreira-Silveetal.[67].Theauthorsob- servedthatsomealcoholdehydrogenasestransformed phenylacetaldoximetotheprimaryalcoholviatheimine andaldehydeintermediates,suggestingthatthehydrideof theco-factorwastransferredtotheN-atomoftheoxime moietyratherthantheC-atom. AlanineracemaseisakeyPLP-dependantenzymein cyclosporine(awellknownimmunosuppressantdrug) biosynthesis.DiSalvoetal.[68]reportedthatjustlike serinehydroxymethyltransferaseandthreoninealdolase, theclonedracemasewasabletocarryoutbothretroal- dolcleavageandtransaminationreactions.Itisastrong evidencethatthesethreeenzymesillustratedivergent evolutionfromacommonancestorwhichperhapssingly performedalltheseindividualreactions.So,thespecia- lisedfunctionsevolvedbuttheenzymesretainedfeatures whichareresponsibleforthepromiscuousbehaviour. AnovelprotocolfortheD-aminoacylasecatalyzed doubleMichaeladditionwasdevelopedbyChenetal. [69].Thereactions,whichwereusedforthesynthesisof (hetero)spiro[5.5]undecanederivativesproducedthe cis isomersinallthecases.Grulichetal.[70]providea usefulsummaryoftheapplicationsofPenicillinGacy- laseswhichareknowntocatalysetransesterifications, MarkonikovadditionsorHenryreactions.Unfortunately, thecatalyticefficiencyreportedsofarinthesepromiscu- ousreactionsisfarfromsatisfactoryforanybiotechno- logicalapplications.However,asNobelietal.[11]had pointedout,suchresultslaythefoundationofsubsequent effortsusingproteinengineering/directedevolutionto improveuponthesecatalyticactivities.Largenumberof successfulresultswhichprovethisarealreadyavailablein theliterature[12,71-73]. Heetal.[74]reportedforthefirsttimethathenegg whitelysozyme(HEWL)efficientlypromotestheone- pot,three-componentaza-Diels-Alderreactionofaro- maticaldehydes,aromaticamineand2-cyclohexen-1-one. Underoptimisedconditions,yieldsupto98%andstereo- selectivityof endo/exo ratiosupto90:10wereobtained. Similarly,Baasetal.[75]havereviewedtheenzyme promiscuityinfivemembersofthetautomerasesuper family.Theseenzymesshowdiversecatalyticactivities encompassingC-H,C-C,C-Oandcarbon-halogenbonds. ThereviewofBaasetal.[75]discusseshowlookingat promiscuousactivitieshelpsinunderstandingofmechan- ismofnormalactivitiesoftheenzymes.Itisinteresting thatallenzymeshaveN-terminalprolineasakeycatalytic residue.Itisverywellestablishedthatsomeaminoacids assuchcatalysediversekindsofreactionsontheirown [76,77].Obviously,thecatalyticpromiscuityofenzymes hasitsorigininconfluenceofdifferentfactors[11].Table2 summarizestheusefulnessofcatalyticpromiscuityinbio- catalysis(Table2). Archaeconstitutetheoldestorganismsonourearth. Giventheharsherconditionsprevalentinthosedays, archaeareprominentexamplesofextremophiles.The specialisedfunctionsoftheenzymes(partoftheevolved metabolism)inmorecomplexorganismhaveevolved fromthesmallnumberofenzymeswhichtheseances- torshad.So,theseorganismsconstitutevaluablesystems totrackextensivepromiscuityshownbytheearlyen- zymes.Jiaetal.havedetailedbothpromiscuityaswellas moonlightingshownbyarchaeenzymes[82].Archaeen- zymesarerichinintrinsicdisorder.Jiaetal.[82]point outthatcapacityforthestructuretosurviveunderharsh conditionsandmultitasking(whichisseenaspromiscu- ityandmoonlighting)requiredconformationalpliability. Itisinterestingthatmany “ hubproteins ” importantin metabolicregulationsinmanyorganismshavebeenfound tobeintrinsicallydisorderedproteins(IDPs)[83].So, promiscuityisonefacetoftheevolutionarydesignofen- zymes.AsSkolnicketal.[84]state,promiscuousbehaviour isthebiochemicalnoise(lowlevel,ligand-proteininterac- tions)whichwasnearlyimpossibletoeliminateasenzyme moleculesevolvedtoassumespecialisedcatalyticroles. Theexampleofglutathionetransferaseisespeciallyin- teresting.Atkinsgroup[85]havediscussedthattwoiso- formsofglutathionetransfereasesinhumansvastly differintheircatalyticpromiscuity.Thishighlightsthe conceptualrelationshipbetweenthephenomenonofiso- enzymestopromiscuousbehaviouraspointedoutbyone ofusfewyearsback[5].TheA-classGSTA1-1showedthe fairlywidesubstratespecificityasadetoxificationenzyme. Ontheotherhand,GSTA4-4acteduponlipidperoxida- tionproducts.Thiswasoneoftheearlyreportstopoint outthat “ conformationalplasticity ” isinbuilttoachieve catalyticpromiscuityatthecostofstability[85].More recentworkfromthesamegroup[86]concludesthat smoothbarrierfreetransitionswithinthelocalconform- ationallandscapeoftheactivesiteisassociatedwiththe morepromiscuousGST.Furthermore,localmoltenglob- ulebehaviouroptimizesthecatalyticfunctionoftheGST indetoxification[87].So,catalyticpromiscuitymaynotbe Arora etal.SustainableChemicalProcesses (2014) 2:25 Page6of9 justaccidental “ noise ” [84]butpartofanintentionalbio- catalyticdesign. Labrou ’ sgroup[88]haverecentlyreportedaGSTfrom A.tumefaciens whichrepresentsanovelclassofbacter- ialGSTsuperfamily.Itsactivesiteisquitedifferentfrom cytosolicGSTreportedsofar.Itmaybeinterestingto seetheunravellingofitsspecificityintheyearstocome. Finally,aspointedoutbythem,GSTsarealsopossibly involvedinthestorageandtransportationofwidevariety ofbiologicalmoleculesandthusarealsomoonlighting proteins.Thisthreewaycorrelationbetweenisoenzymes, promiscuousenzymesandmoonlightingproteinshave beenpointedoutearlier[5].Tosumup,theconform- ationalpliabilitymaybelocalorglobalinproteinstruc- tureasthecausebehindpromiscuity. Conclusions Fewtrendsareclear.Whileduringearlyfewyears,ex- amplesofcatalyticpromiscuityweremostlyconcerned withapplicationsoflipases;lastfewyearshaveseen otherclassesofenzymes(otherhydrolases,oxidoreduc- tases,transferasesetc.)beingequallycapableofshowing catalyticpromiscuity. Theshiftfromourbeliefinenzymespecificitytothe realizationthatthesebiocatalystsarefairlypromiscuous hasnotbeengradual.Somekeymilestonescanbeidenti- fied.Weacceptedtheideaofbroadspecificity(latelycalled relaxedspecificity)longago.Isoformsorisoenzymes,the enzymesfromthesameorganism,carryingsimilarcatalytic activitybutwithdifferentspecificityandkineticbehaviour havebeenagainknownsinceseveraldecades[5].Theca- talysisstartswithmolecularrecognitionofthe “ substrate ” bytheenzyme.Thebindingsite,partoftheactivecentre wasknowntoshowpromiscuousbehaviourwhentex- tiledyesemergedaspowerfulaffinityligands[89].The catalyticpromiscuitylargelyarisesbecausemanydif- ferent “ substrates ” caninteractwithdifferentsidechains ofaminoacidstoresultindifferenttransitionstates. Table2Applicationsofcatalyticpromiscuityforusefulbiotransformations BiotransformationsSubstrate(s)Enzyme(s)References Regio-andstereoselective reductions DiketonesADHsfrom Lactobacilluskefir,Rhodococcus ruber,Thermoanaerobacter etc. [ 66 ] ReductionreactionPhenylacetaldoximeADHsfrom Rhodococcusruber,Ralstoniasp., Thermoanaerobacter etc. [ 67 ] Aza-Diels-Alderreaction4-chlorobenzaldehyde,cyclohexenone,4-anisidineHeneggwhitelysozyme(HEWL)[ 74 ] DominoKnoevenagel/ intramoleculartransesterification Salicylaldehyde,ethylacetoacetateAlkalineproteasefrom Bacillus licheniformis (BLAP) [ 58 ] TransesterificationreactionGuaifenesin,vinylacetatePenicillinGacylase[ 78 ] KnoevenagelreactionAromaticaldehyde,acetylacetone/EthylacetoacetatePapainfrom Caricapapaya latex[ 54 ] Ugireactionforpeptide synthesis(MCR) * Aldehyde,amine,isocyanideNovozyme435(commerciallyavailable, immobilized Candidaantarctica lipaseB) [ 47 ] AsymmetricaldolreactionAromaticaldehyde,cyclicketoneNucleasep1from Penicilliumcitrinum [ 51 ] AsymmetricaldolreactionHeterocyclicketone,aromaticaldehydeFicinfromfigtreelatex[ 53 ] Synthesisof4-thiazolidinonesAromaticaldehyde,benzylamine,mercaptoaceticacidTrypsinfromporcinepancreas[ 55 ] Dominooxa-Michael/aldol condensations Salicylaldehyde,methylvinylketone
-amylasefrom Bacillussubtilis [ 59 ] Henryreaction4-cyanobenzaldehyde,nitromethaneGlucoamylasefrom Aspergillusniger (AnGA)[ 60 ] Retroaldolandtransamination reactions L-threonineandL- allo -threonine(forretroaldol reaction);D-andL-alanine(fortransaminationreaction) Alanineracemasefrom Tolypocladium inflatum [ 68 ] DoubleMichaeladditionreactionCyclohexane-1,3-dioneand(1E,4E)-1, 5-diphenylpenta-1,4-dien-3-one D-aminoacylase[ 69 ] EnantioselectivealdolreactionIsatinderivatives,cyclicketonesNucleasep1from Penicilliumcitrinum [ 50 ] Synthesisof2,4-disubstituted thiazoles(MCR) * Benzylamine,isobutyraldehyde,thioaceticacid, methyl3-(dimethylamino)-2-isocyanoacrylate Porcinepancreaticlipase(PPL)[ 44 ] Michaeladdition-cyclizationcas- cadereaction Substitutedbenzalacetonesand1,3-cyclicdiketones E.coli BioHesterase[ 52 ] Synthesisofsubstituted 2H-chromemes(MCR) * Salicylaldehyde,acetophenone,methanolPorcinepancreaticlipase[ 79 ] Baylis-Hillmanreaction p -nitrobenzaldehydeandmethylvinylketone E.coli BioHesterase[ 80 ] Biginellireaction(MCR) * Urea,ethylacetoacetate,vinylacetateTrypsinfromporcinepancreas[ 81 ] *standsforMulti-componentreaction. Arora etal.SustainableChemicalProcesses (2014) 2:25 Page7of9 Thisisakintoageneralpractitionerbecomingaspe- cialistinmedicalsciencebutretainingenoughknowledge ofhowtotreatmanydiseases.Inthebeginning,wehad RNAworld[90].Thencameearlyenzymes.Ascomplex metabolismswererequiredwithevolutionofmorecom- plexorganisms,morespecialisedenzymesemerged.These enzymesdidnotforgetentirelywhattheirancestorswere capableof. Whitebiotechnologycandefinitelyprofitbyexploiting manyofthecatalyticpowerswhichenzymesdidnoten- tirely “ forget ” !Thisoverviewhaslookedattheproverbial “ tipoftheiceberg ” .Hopefully,itwilldrawattentionof manybiotechnologiststolookatthehugeicebergofpo- tentialapplicationofthesegreencatalyststonurture sustainableapproachesinchemicalindustries. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions BAplayedalargeroleinthesearchforthecurrentliterature.Allauthors participatedindraftingofthetext,readandapprovedthefinalmanuscript. 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