Microbial transformation, or biotransformation, refers to processes where microorganisms convert organic compounds into structurally related products through one or few enzymatic reactions, as opposed to fermentation which involves many reactions. Biotransformations are preferred over chemical reactions for reasons such as substrate specificity, stereospecificity, and producing little environmental pollution. Various types of chemical reactions occur in biotransformations including oxidation, reduction, hydrolysis, and isomerization. A wide variety of biological catalysts can be used including growing cells, resting cells, immobilized cells, cell-free extracts, and immobilized enzymes. Product recovery methods include precipitation, extraction, adsorption, and distillation.

1. Microbial transformation
By: Bijaya Kumar Uprety
1

2. Introduction
•Biotransformations(bioconversionormicrobialtransformation)referstotheprocessesinwhichmicroorganismsconvertorganiccompoundsintostructurallyrelatedproducts.
•Inotherword,biotransformationdealswithmicrobial(enzymatic)conversionofasubstrateintoaproductwithlimitednumber(oneorfew) enzymaticreactions.
•Thisisincontrasttofermentationwhichinvolvesalargenumberreactions.
2

3. •Thesignificanceofbioconversionreactionsbecomesobviouswhentheproductionofaparticularcompoundiseitherdifficultorcostlybychemicalmethods.
•Furtherbiotransformationsaregenerallypreferredtochemicalreactionsbecauseofsubstratespecificity, stereospecificity,andmixedreactionconditions(pH, temperature,pressure).
•Theenvironmentalpollutionduetobiotransformationisalmostinsignificantornegligible.
•Inaddition,itiseasytoapplyrecombinantDNAtechnologytomakedesiredimprovementsinbiotransformations.
•Anotheradvantageofbiotransformationsisthatitiseasytoscaleuptheprocessduetolimitednumberofreactions.
3

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. •Manyatimesbiotransformationinvolvesmorethanonetypeofreactions.
•Theconversiontimerequiredforbiotransformationisrelatedtothetypeofreaction,thesubstrateconcentrationandthemo’sused.
•Ingeneraloxidation,hydrolysisanddehydrationreactionsarecompletedinfewhours.
5

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. Growing cells
•Thedesiredcellsarecultivatedinasuitablemedium.
•Asthegrowthofthecellsoccurs(6-24hours),aconcentratedsubstrateisaddedtotheculture.
•Sometimes,additionofemulsifiers(Tween,organicsolvents)isrequiredtosolubilizesubstratesand/orproductseg.Steroidbiotrasformation.
•Thesubstrateconversiontoproductcanbemonitoredbyspectroscopicorchromatographictechniques.
•Biotransformationcanbeterminatedwhentheproductformationisoptimum.
7

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. Immobilized cells
•Biotransformationcanbecarriedoutcontinuouslybyemployingimmobilizedcells.
•Further,thesamecellscouldbeusedfornumeroustime.
•Severalbioconversionwithsingleormultistagereactionareinfactcarriedoutbyusingimmobilizedcells.Eg.commercialproductionofL-analineandmalicacid.
9

10. Immobilized enzymes
•Cell-freeenzymesystemsintheformofimmobilizedenzymesaremostcommonlyusedinbiotransformation,duetofollowingreasons:
Noundesirablesidereaction.
Desiredproductsarenotdegraded.
Notransportbarrieracrossthecellmembraneforthesubstrateorproduct.
Isolationandrecoveryoftheproductissimplerandeasier.
Eg.Glucoseisomerase,penicillinacylase.
10

11. Product recovery in biotransformation
•Inmostbiotrasformationreactions,thedesiredendproductsareextracellular.
•Theproductmaybeeitherinasolubleorsuspendedstate.
•Whenwholecellsareused,theyhavetobeseparatedandrepeatedlywashedwithwaterororganicsolventasrequired.
•Theextractedproductcanberecoveredbyemployingthecommonlyusedtechniques-precipitationbysalts, extractionwithsolvents,adsorptiontoion-exchangers, etc.
•Volatileproductscouldberecoveredbydirectdistillationfromthemedium.
11

12. Biotransformation of steroids
Asteroidisatypeoforganiccompoundthatcontainsacharacteristicarrangementoffourcycloalkaneringsthatarejoinedtoeachother.
12

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

15. 15

16. 16

17. 17

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

19. 19

20. Methodologies for biotransformation
•Avarietyofsubstances,namely:growingcultures,restingcells,immobilizedcells, spores,enzymes,andimmobilizedenzymesystemsmaybeemployedoverwhelminglyinthemicrobialbiotransformationofaplethoraoforganiccompounds.
•Afewspecificmethodologiesinvolvinggrowingcultures,restingcells,andimmobilizedcellsshallbediscussedindividuallyinthenextslides:
20

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. 2.RestingCells:
•Insuchcriticalsituationswhentheenzymeinductionaffordedbytheaddedsubstrateisnotquitenecessaryandurgent, restingcellsmaybeemployedprofuselyandeffectively.
•However,therestingcellsdoofferatremendousadvantagewherebythegrowthinhibitionbythesubstrateiseliminatedcompletely.Besides,thepresenceofhigh-celldensitiesthatessentiallypromoteanenhancedlevelofproductivitymaybeemployed;simultaneously,theveryriskofanypossiblescopeofcontaminationisminimisedappreciably.
•Interestingly,thereareseveralbiotransformationreactionsthatexclusivelyandpredominantlytakeplaceinthe‘buffersolution’andthiseventuallyrenderstheultimaterecoveryofthe‘desiredproduct’relativelyeasyandconvenient.
22

23. 3.ImmobilizedCells
•Inmorerecenttimes,ahostofbiotransformationmethodologiesdomakeuseoftheimmobilizedcellsthusaffordingthebiggestevenadvantageouspluspointthattheprocesscouldbecarriedoutsimultaneously;besides,thecellsmightbeemployedoverandoveragain.
Applications:Inactualpractice,theimmobilizedbacterialcellsthatinvariablycatalyzeeithersingle-stagereactionormulti-stagereaction,arepresentlyexploitedinthelarge-scaleproductionofL-alanine,asparticacid,andmalicacid.
23

24. Selection of organism
•Theselectionofstrainseitherfromitsnaturalsourcesorfromthevariousavailablecultureswhicharesolelyresponsibleforcatalyzingthedesiredbiotransformationreaction(s)isnotonlyvitalandcriticalbutalsoofgreatimportance.
•Ithasbeenobservedthattherearequiteafewmicroorganismsthatusuallycarryoutthedesiredbioconversionswiththehelpofarelatedchemicalentity.
•Insteroidonemayencounteraratherdifficultproblemduetothelackofselectivemethodssoastoidentifythecoloniespreciselywhichusuallyperformtheappropriatespecificactivity.
24

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. •Besides,theresultingintermediatesmaygetaccumulated, whereasthelesion-bearingmutantscanbeisolatedconveniently.Furthermore,mutantsmayalsobeisolatedwhichareincapableofaccumulatingan‘undesirablecompound’.
26

27. 2.FiltrationEnrichmentMethod:
•Inthiscase,aftermutagenesisthesporesoffilamentousorganismse.g.,actinomycetes,fungi,aremadetodevelopinaliquidminimalmedium.
•Themicrocoloniesofprototrophsthusdevelopedaremeticulouslyseparatedbyfiltration,wherebythesporesofauxotrophsthatwereunabletogrowleftbehindinthefiltrate.
•Thefiltrateobtainedinthismannerinsubsequentlyplatedandtheresultingcoloniesareadequatelycheckedforauxotrophiccharacteristics.
27

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. 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

30. 30