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Microbial transformation 
By: Bijaya Kumar Uprety 
1
Introduction 
•Biotransformations(bioconversionormicrobialtransformation)referstotheprocessesinwhichmicroorganismsconverto...
•Thesignificanceofbioconversionreactionsbecomesobviouswhentheproductionofaparticularcompoundiseitherdifficultorcostlybyche...
Types of Biotransformation reactions 
•Many types of chemical reactions occur in biotransformations. 
•These include oxida...
•Manyatimesbiotransformationinvolvesmorethanonetypeofreactions. 
•Theconversiontimerequiredforbiotransformationisrelatedto...
Sources of Biocatalysts and techniques for biotransformation 
•A wide variety of biological catalysts can be used for biot...
Growing cells 
•Thedesiredcellsarecultivatedinasuitablemedium. 
•Asthegrowthofthecellsoccurs(6-24hours),aconcentratedsubst...
Non-growing cells 
•These are preferred for biotransformation rxndue to following reasons: 
Very high concentration of su...
Immobilized cells 
•Biotransformationcanbecarriedoutcontinuouslybyemployingimmobilizedcells. 
•Further,thesamecellscouldbe...
Immobilized enzymes 
•Cell-freeenzymesystemsintheformofimmobilizedenzymesaremostcommonlyusedinbiotransformation,duetofollo...
Product recovery in biotransformation 
•Inmostbiotrasformationreactions,thedesiredendproductsareextracellular. 
•Theproduc...
Biotransformation of steroids 
Asteroidisatypeoforganiccompoundthatcontainsacharacteristicarrangementoffourcycloalkanering...
Design of biotransformation process 
•Ithasbeenadequatelyobservedthatthemostcrucialandpivotalbiotransformationprocessesare...
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15
16
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•Inadditiontotheaboveremarkableexplicitexamplesithasbeenamplydemonstratedandadequatelysubstantiatedscientificallythat‘plan...
19
Methodologies for biotransformation 
•Avarietyofsubstances,namely:growingcultures,restingcells,immobilizedcells, spores,en...
1.GrowingCultures 
•Themethodologiesthatareintimatelyassociatedwithgrowingculturesessentiallyinvolvethestrainthatarecultiv...
2.RestingCells: 
•Insuchcriticalsituationswhentheenzymeinductionaffordedbytheaddedsubstrateisnotquitenecessaryandurgent, r...
3.ImmobilizedCells 
•Inmorerecenttimes,ahostofbiotransformationmethodologiesdomakeuseoftheimmobilizedcellsthusaffordingthe...
Selection of organism 
•Theselectionofstrainseitherfromitsnaturalsourcesorfromthevariousavailablecultureswhicharesolelyres...
1.ModifiedEnrichmentMethod: 
•Themodifiedenrichmentmethodisinvariablyusedfortheisolationofmutantsblockedinthesubstratediss...
•Besides,theresultingintermediatesmaygetaccumulated, whereasthelesion-bearingmutantscanbeisolatedconveniently.Furthermore,...
2.FiltrationEnrichmentMethod: 
•Inthiscase,aftermutagenesisthesporesoffilamentousorganismse.g.,actinomycetes,fungi,aremade...
3.Penicillin-SelectionProcedure: 
•Inpenicillin-selectionproceduretheprevailinggrowingcellsarekilledselectivelybythe‘antib...
5.SprayingwithReagents(orIncorporatingIndicatorDyes): 
Onemayobserveeitherthepresenceorabsenceofspecificenzymeactivitiesal...
30
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Microbial transformation

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Microbial transformation

  1. 1. Microbial transformation By: Bijaya Kumar Uprety 1
  2. 2. Introduction •Biotransformations(bioconversionormicrobialtransformation)referstotheprocessesinwhichmicroorganismsconvertorganiccompoundsintostructurallyrelatedproducts. •Inotherword,biotransformationdealswithmicrobial(enzymatic)conversionofasubstrateintoaproductwithlimitednumber(oneorfew) enzymaticreactions. •Thisisincontrasttofermentationwhichinvolvesalargenumberreactions. 2
  3. 3. •Thesignificanceofbioconversionreactionsbecomesobviouswhentheproductionofaparticularcompoundiseitherdifficultorcostlybychemicalmethods. •Furtherbiotransformationsaregenerallypreferredtochemicalreactionsbecauseofsubstratespecificity, stereospecificity,andmixedreactionconditions(pH, temperature,pressure). •Theenvironmentalpollutionduetobiotransformationisalmostinsignificantornegligible. •Inaddition,itiseasytoapplyrecombinantDNAtechnologytomakedesiredimprovementsinbiotransformations. •Anotheradvantageofbiotransformationsisthatitiseasytoscaleuptheprocessduetolimitednumberofreactions. 3
  4. 4. Types of Biotransformation reactions •Many types of chemical reactions occur in biotransformations. •These include oxidation, reduction, hydrolysis, condensation, isomerization, formation of new C-C bonds, synthesis of chiral compounds and reversal of hydrolytic reactions. •Among these, oxidation, isomerisation and hydrolysis reactions are more commonly obser 4
  5. 5. •Manyatimesbiotransformationinvolvesmorethanonetypeofreactions. •Theconversiontimerequiredforbiotransformationisrelatedtothetypeofreaction,thesubstrateconcentrationandthemo’sused. •Ingeneraloxidation,hydrolysisanddehydrationreactionsarecompletedinfewhours. 5
  6. 6. Sources of Biocatalysts and techniques for biotransformation •A wide variety of biological catalysts can be used for biotransformation reactions. •Includes: Growing cells, Resting cells, Killed cells, Immobilized cells, Cell-free extract, Enzymes and Immobilized enzymes. 6
  7. 7. Growing cells •Thedesiredcellsarecultivatedinasuitablemedium. •Asthegrowthofthecellsoccurs(6-24hours),aconcentratedsubstrateisaddedtotheculture. •Sometimes,additionofemulsifiers(Tween,organicsolvents)isrequiredtosolubilizesubstratesand/orproductseg.Steroidbiotrasformation. •Thesubstrateconversiontoproductcanbemonitoredbyspectroscopicorchromatographictechniques. •Biotransformationcanbeterminatedwhentheproductformationisoptimum. 7
  8. 8. Non-growing cells •These are preferred for biotransformation rxndue to following reasons: Very high concentration of substrate can be used ( high concgrowth of cells stops usually) Cells can be washed and used thus there will be no contaminating substances. Conversion efficiency of substrate to product is high. Biotransformation can be optimisedby creating suitable environmental condition (egpH, temp). Product isolation and its recovery is easy. 8
  9. 9. Immobilized cells •Biotransformationcanbecarriedoutcontinuouslybyemployingimmobilizedcells. •Further,thesamecellscouldbeusedfornumeroustime. •Severalbioconversionwithsingleormultistagereactionareinfactcarriedoutbyusingimmobilizedcells.Eg.commercialproductionofL-analineandmalicacid. 9
  10. 10. Immobilized enzymes •Cell-freeenzymesystemsintheformofimmobilizedenzymesaremostcommonlyusedinbiotransformation,duetofollowingreasons: Noundesirablesidereaction. Desiredproductsarenotdegraded. Notransportbarrieracrossthecellmembraneforthesubstrateorproduct. Isolationandrecoveryoftheproductissimplerandeasier. Eg.Glucoseisomerase,penicillinacylase. 10
  11. 11. Product recovery in biotransformation •Inmostbiotrasformationreactions,thedesiredendproductsareextracellular. •Theproductmaybeeitherinasolubleorsuspendedstate. •Whenwholecellsareused,theyhavetobeseparatedandrepeatedlywashedwithwaterororganicsolventasrequired. •Theextractedproductcanberecoveredbyemployingthecommonlyusedtechniques-precipitationbysalts, extractionwithsolvents,adsorptiontoion-exchangers, etc. •Volatileproductscouldberecoveredbydirectdistillationfromthemedium. 11
  12. 12. Biotransformation of steroids Asteroidisatypeoforganiccompoundthatcontainsacharacteristicarrangementoffourcycloalkaneringsthatarejoinedtoeachother. 12
  13. 13. Design of biotransformation process •Ithasbeenadequatelyobservedthatthemostcrucialandpivotalbiotransformationprocessesaredesignedandbaseduponavarietyofchemicalreactionswhichmaybeclassifiedunderseveralcategories,suchas:(a)oxidation;(b)reduction;(c)hydrolysis;(d)condensation;(e) isomerization;(f)formationofnewerC–Cbonds;and(h)introductionofheterofunctionalmoieties. •Ingeneral,thevariouskindsofbiotransformationprocessesinvolvingtypicalchemicalreactionsalongwithcertainspecificexamplesandthepercentageefficiencyofconversionaresummarizedinthefollowing.Apossibleexplanationofthereaction(s)involvedhasbeenincludedinordertohaveabetterunderstandingofthesechemicalpathways. •Biotransformationdesignshavebeenaccomplishedwithtremendoussuccessforalargenumberofcompounds,namely:cardiacglycoside‘digoxin’,acetyltropine,benzylisoquinolineetc.Sofar,thevarioustypicalexamplesthathavebeencitedinthebelowtableareexclusivelyrelatedtoavarietyofchemicalreactionsinthepresenceofmicroorganisms. 13
  14. 14. 14
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  18. 18. •Inadditiontotheaboveremarkableexplicitexamplesithasbeenamplydemonstratedandadequatelysubstantiatedscientificallythat‘plantcells’arealsocapableoftransformingawiderangeofsubstrates;and, therefore,carryoutalargenumberofreaction(s),forinstance:oxidation, hydroxylation,reduction,methylation,glucosylation,acetylation, aminoacylationandthelike. •Forexample: 1.GlycosylationofsalicylicacidbytheculturesofMallotusjaponicayieldsaproductthatpossessesanappreciablehighanalgesicactivity,andalsoexhibitsexcellentbettertoleranceinthestomachincomparisontoacetylsalicylicacid(i.e.,aspirin). 2.TransformationofSteviol(aglucon)intoStevioside(glucoside):ThetransformationofSteviol(i.e.,hydroxydehydrostevicacid)bythecellsofSteviarebaudiana(Bert.)Hemsl.(EupatoriumrebaudianumBert.) Compositae,alsocalledyerbadulce(Habitat:Paraguay),intoaglucosideknownassteviosidewhichisprovedtobe300timessweeterthansucrose,andhenceusedasasweetner. 18
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  20. 20. Methodologies for biotransformation •Avarietyofsubstances,namely:growingcultures,restingcells,immobilizedcells, spores,enzymes,andimmobilizedenzymesystemsmaybeemployedoverwhelminglyinthemicrobialbiotransformationofaplethoraoforganiccompounds. •Afewspecificmethodologiesinvolvinggrowingcultures,restingcells,andimmobilizedcellsshallbediscussedindividuallyinthenextslides: 20
  21. 21. 1.GrowingCultures •Themethodologiesthatareintimatelyassociatedwithgrowingculturesessentiallyinvolvethestrainthatarecultivatedinanappropriateculturemedium,andsubsequentlyaconcentratedsubstratedsolutionisusuallyincorporatedafteranappreciablegrowthofthecultureafteralapseof6to24hours.Afewnoteworthyvariantsofthisparticularprocedureareasstatedbelow: (a)Usageofarelativelyverylargeinoculum, (b)Incorporatingtheconcentratedsubstrateimmediatelywithoutpermitting,agrowthphasetocommence, (c)Usageof‘emulsifiers’e.g.,Tweens(i.e.,Tween-20,40,60,80— syntheticsurfectants)orwater-misciblesolventse.g.,acetone,ethanol, dimethylformamide(DMF),dimethylsulphoxide(DMSO)maybeemployedtoaidthedissolutionofrathersparinglysolublesubstancesquiteconveniently. 21
  22. 22. 2.RestingCells: •Insuchcriticalsituationswhentheenzymeinductionaffordedbytheaddedsubstrateisnotquitenecessaryandurgent, restingcellsmaybeemployedprofuselyandeffectively. •However,therestingcellsdoofferatremendousadvantagewherebythegrowthinhibitionbythesubstrateiseliminatedcompletely.Besides,thepresenceofhigh-celldensitiesthatessentiallypromoteanenhancedlevelofproductivitymaybeemployed;simultaneously,theveryriskofanypossiblescopeofcontaminationisminimisedappreciably. •Interestingly,thereareseveralbiotransformationreactionsthatexclusivelyandpredominantlytakeplaceinthe‘buffersolution’andthiseventuallyrenderstheultimaterecoveryofthe‘desiredproduct’relativelyeasyandconvenient. 22
  23. 23. 3.ImmobilizedCells •Inmorerecenttimes,ahostofbiotransformationmethodologiesdomakeuseoftheimmobilizedcellsthusaffordingthebiggestevenadvantageouspluspointthattheprocesscouldbecarriedoutsimultaneously;besides,thecellsmightbeemployedoverandoveragain. Applications:Inactualpractice,theimmobilizedbacterialcellsthatinvariablycatalyzeeithersingle-stagereactionormulti-stagereaction,arepresentlyexploitedinthelarge-scaleproductionofL-alanine,asparticacid,andmalicacid. 23
  24. 24. Selection of organism •Theselectionofstrainseitherfromitsnaturalsourcesorfromthevariousavailablecultureswhicharesolelyresponsibleforcatalyzingthedesiredbiotransformationreaction(s)isnotonlyvitalandcriticalbutalsoofgreatimportance. •Ithasbeenobservedthattherearequiteafewmicroorganismsthatusuallycarryoutthedesiredbioconversionswiththehelpofarelatedchemicalentity. •Insteroidonemayencounteraratherdifficultproblemduetothelackofselectivemethodssoastoidentifythecoloniespreciselywhichusuallyperformtheappropriatespecificactivity. 24
  25. 25. 1.ModifiedEnrichmentMethod: •Themodifiedenrichmentmethodisinvariablyusedfortheisolationofmutantsblockedinthesubstratedissimilationmechanism. •Inthisspecificinstance,asteroidsubstrateisnormallyincorporatedasthesoleC-sourceexclusivelyina‘minimalmedium’seededadequatelywiththesoildilutions. •Thecellsthatcausesthedegradationofthesubstratewillultimatelygrow;andare,therefore,subsequentlytransferredtothesamemediumbutparticularlyenrichedwithanotherC-source,forinstance:glucose. •However,themutantsmaybepresentwhicharestrategicallyblockedatdifferentstagesintheprocessofdegradationofthesteroidsubstrate,butmayconsumeglucoseastheC-source. •Ithasbeenprofuselyestablishedandreportedthatafairlylargenumberofmicrobialstrainsviz.,eubacteria,yeasts,molds,andstreptomycetesmaybestoredandmaintainedstrictlyaspertherecommended‘standardmethods’,suchas:agarslant,soilculture,frozenculture,andlypholizedculturepreservedattemperaturesrangingbetween–20°Cto–170°C. 25
  26. 26. •Besides,theresultingintermediatesmaygetaccumulated, whereasthelesion-bearingmutantscanbeisolatedconveniently.Furthermore,mutantsmayalsobeisolatedwhichareincapableofaccumulatingan‘undesirablecompound’. 26
  27. 27. 2.FiltrationEnrichmentMethod: •Inthiscase,aftermutagenesisthesporesoffilamentousorganismse.g.,actinomycetes,fungi,aremadetodevelopinaliquidminimalmedium. •Themicrocoloniesofprototrophsthusdevelopedaremeticulouslyseparatedbyfiltration,wherebythesporesofauxotrophsthatwereunabletogrowleftbehindinthefiltrate. •Thefiltrateobtainedinthismannerinsubsequentlyplatedandtheresultingcoloniesareadequatelycheckedforauxotrophiccharacteristics. 27
  28. 28. 3.Penicillin-SelectionProcedure: •Inpenicillin-selectionproceduretheprevailinggrowingcellsarekilledselectivelybythe‘antibiotic’treatment,therebyenrichingtheauxotrophsthatareincapableofgrowinguponthe‘minimalmedium’. •Thus,exclusivelybasedupontheirmodeofactionaplethoraof‘inhibitors’otherthanpenicillinmayalsobeemployedeffectivelyinthisprocedure, namelydihydrostreptomycinforPseudomonasaeruginosa;nystalinforHansenulapolymorpha,Penicilliumchrysogenum,Aspergillusnidulans, andSaccharomycescerevisiae;nalidixacidforSalmonellatyphimurium; colistinforthepenicillin-resistantHydrogenomonasstrainH16. 4.SodiumPentachlorophenolate:Thesaltsodiumpentachlorophenolatealsoaffordsenrichmentprocedurebyvirtueofitsgreatertoxicityparticularlyagainstthe‘germinatingspores’incomparisontothe‘vegetativecells’. Example:Theabovemethodhasbeensuccessfullyappliedwithseveralorganisms,suchas:Penicilliumchrysogenum;Streptomycesaureofaciens; Streptomycesolivaceus;andBacillussubtilis. 28
  29. 29. 5.SprayingwithReagents(orIncorporatingIndicatorDyes): Onemayobserveeitherthepresenceorabsenceofspecificenzymeactivitiesalmostdirectlyinthecoloniesthatareallowedtogrownonplatesbyemployingeitherofthetwoavailablecommonprocedures,namely:(a) sprayingwithappropriatereagents;and(b)incorporatingindicator-dyesrightintotheculturemedium. 6.InhibitionofAssayOrganisms:Inthisspecificinstancetheantibiotically- activecompoundsmaybedetectedquiteeasilyandconvenientlybymeasuringtheinhibitionofsensitiveassayorganisms.Thisprocedureallowstheprecisedetermination(assay)ofthe‘antibioticcontent’ofanunknown solutionusingareferencestandardsimultaneously. 7.AgarPlugMethod:Theagarplugmethodisregardedtobeoneofthemostreliableandprecisetechniqueswhereintheagarcylindershaving‘single-colonies’aretransferredtotestplatesafterdueincubationpreferablyinamoistchamberasdepictedinfiguregivenbelow: 29
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