Microbial transformation

1. Microbial
transformation
By: Bijaya Kumar Uprety
1

2. Introduction
•Biotransformations(bioconversionormicrobial
transformation)referstotheprocessesinwhich
microorganismsconvertorganiccompoundsinto
structurallyrelatedproducts.
•Inotherword,biotransformationdealswith
microbial(enzymatic)conversionofasubstrateinto
aproductwithlimitednumber(oneorfew)
enzymaticreactions.
•Thisisincontrasttofermentationwhichinvolvesa
largenumberreactions.
2

3. •Thesignificanceofbioconversionreactionsbecomesobvious
whentheproductionofaparticularcompoundiseither
difficultorcostlybychemicalmethods.
•Furtherbiotransformationsaregenerallypreferredto
chemicalreactionsbecauseofsubstratespecificity,
stereospecificity,andmixedreactionconditions(pH,
temperature,pressure).
•Theenvironmentalpollutionduetobiotransformationis
almostinsignificantornegligible.
•Inaddition,itiseasytoapplyrecombinantDNAtechnologyto
makedesiredimprovementsinbiotransformations.
•Anotheradvantageofbiotransformationsisthatitiseasyto
scaleuptheprocessduetolimitednumberofreactions.
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. •Manyatimesbiotransformationinvolvesmorethanonetype
ofreactions.
•Theconversiontimerequiredforbiotransformationisrelated
tothetypeofreaction,thesubstrateconcentrationandthe
mo’sused.
•Ingeneraloxidation,hydrolysisanddehydrationreactionsare
completedinfewhours.
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),a
concentratedsubstrateisaddedtotheculture.
•Sometimes,additionofemulsifiers(Tween,organic
solvents)isrequiredtosolubilizesubstratesand/or
productseg.Steroidbiotrasformation.
•Thesubstrateconversiontoproductcanbe
monitoredbyspectroscopicorchromatographic
techniques.
•Biotransformationcanbeterminatedwhenthe
productformationisoptimum.
7

8. Non-growing cells
•These are preferred for biotransformation rxn
due 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
•Biotransformationcanbecarriedout
continuouslybyemployingimmobilizedcells.
•Further,thesamecellscouldbeusedfor
numeroustime.
•Severalbioconversionwithsingleor
multistagereactionareinfactcarriedoutby
usingimmobilizedcells.Eg.commercial
productionofL-analineandmalicacid.
9

10. Immobilized enzymes
•Cell-freeenzymesystemsintheformof
immobilizedenzymesaremostcommonlyusedin
biotransformation,duetofollowingreasons:
Noundesirablesidereaction.
Desiredproductsarenotdegraded.
Notransportbarrieracrossthecellmembrane
forthesubstrateorproduct.
Isolationandrecoveryoftheproductissimpler
andeasier.
Eg.Glucoseisomerase,penicillinacylase.
10

11. Product recovery in biotransformation
•Inmostbiotrasformationreactions,thedesiredend
productsareextracellular.
•Theproductmaybeeitherinasolubleorsuspended
state.
•Whenwholecellsareused,theyhavetobeseparated
andrepeatedlywashedwithwaterororganicsolvent
asrequired.
•Theextractedproductcanberecoveredbyemploying
thecommonlyusedtechniques-precipitationbysalts,
extractionwithsolvents,adsorptiontoion-exchangers,
etc.
•Volatileproductscouldberecoveredbydirect
distillationfromthemedium.
11

12. Biotransformation of steroids
Asteroidisatypeoforganic
compoundthatcontainsacharacteristic
arrangementoffourcycloalkaneringsthat
arejoinedtoeachother.
12

13. Design of biotransformation process
•Ithasbeenadequatelyobservedthatthemostcrucialandpivotal
biotransformationprocessesaredesignedandbaseduponavarietyof
chemicalreactionswhichmaybeclassifiedunderseveralcategories,such
as:(a)oxidation;(b)reduction;(c)hydrolysis;(d)condensation;(e)
isomerization;(f)formationofnewerC–Cbonds;and(h)introductionof
heterofunctionalmoieties.
•Ingeneral,thevariouskindsofbiotransformationprocessesinvolving
typicalchemicalreactionsalongwithcertainspecificexamplesandthe
percentageefficiencyofconversionaresummarizedinthefollowing.A
possibleexplanationofthereaction(s)involvedhasbeenincludedinorder
tohaveabetterunderstandingofthesechemicalpathways.
•Biotransformationdesignshavebeenaccomplishedwithtremendous
successforalargenumberofcompounds,namely:cardiacglycoside
‘digoxin’,acetyltropine,benzylisoquinolineetc.Sofar,thevarioustypical
examplesthathavebeencitedinthebelowtableareexclusivelyrelatedto
avarietyofchemicalreactionsinthepresenceofmicroorganisms.
13

14. 14

15. 15

16. 16

17. 17

18. •Inadditiontotheaboveremarkableexplicitexamplesithasbeenamply
demonstratedandadequatelysubstantiatedscientificallythat‘plantcells’
arealsocapableoftransformingawiderangeofsubstrates;and,
therefore,carryoutalargenumberofreaction(s),forinstance:oxidation,
hydroxylation,reduction,methylation,glucosylation,acetylation,
aminoacylationandthelike.
•Forexample:
1.GlycosylationofsalicylicacidbytheculturesofMallotusjaponicayieldsa
productthatpossessesanappreciablehighanalgesicactivity,andalso
exhibitsexcellentbettertoleranceinthestomachincomparisonto
acetylsalicylicacid(i.e.,aspirin).
2.TransformationofSteviol(aglucon)intoStevioside(glucoside):The
transformationofSteviol(i.e.,hydroxydehydrostevicacid)bythecellsof
Steviarebaudiana(Bert.)Hemsl.(EupatoriumrebaudianumBert.)
Compositae,alsocalledyerbadulce(Habitat:Paraguay),intoaglucoside
knownassteviosidewhichisprovedtobe300timessweeterthan
sucrose,andhenceusedasasweetner.
18

19. 19

20. Methodologies for biotransformation
•Avarietyofsubstances,namely:growing
cultures,restingcells,immobilizedcells,
spores,enzymes,andimmobilizedenzyme
systemsmaybeemployedoverwhelminglyin
themicrobialbiotransformationofaplethora
oforganiccompounds.
•Afewspecificmethodologiesinvolving
growingcultures,restingcells,and
immobilizedcellsshallbediscussed
individuallyinthenextslides:
20

21. 1.GrowingCultures
•Themethodologiesthatareintimatelyassociatedwithgrowingcultures
essentiallyinvolvethestrainthatarecultivatedinanappropriateculture
medium,andsubsequentlyaconcentratedsubstratedsolutionisusually
incorporatedafteranappreciablegrowthofthecultureafteralapseof6
to24hours.Afewnoteworthyvariantsofthisparticularprocedureareas
statedbelow:
(a)Usageofarelativelyverylargeinoculum,
(b)Incorporatingtheconcentratedsubstrateimmediatelywithout
permitting,agrowthphasetocommence,
(c)Usageof‘emulsifiers’e.g.,Tweens(i.e.,Tween-20,40,60,80—
syntheticsurfectants)orwater-misciblesolventse.g.,acetone,ethanol,
dimethylformamide(DMF),dimethylsulphoxide(DMSO)maybe
employedtoaidthedissolutionofrathersparinglysolublesubstances
quiteconveniently.
21

22. 2.RestingCells:
•Insuchcriticalsituationswhentheenzymeinductionafforded
bytheaddedsubstrateisnotquitenecessaryandurgent,
restingcellsmaybeemployedprofuselyandeffectively.
•However,therestingcellsdoofferatremendousadvantage
wherebythegrowthinhibitionbythesubstrateiseliminated
completely.Besides,thepresenceofhigh-celldensitiesthat
essentiallypromoteanenhancedlevelofproductivitymaybe
employed;simultaneously,theveryriskofanypossiblescope
ofcontaminationisminimisedappreciably.
•Interestingly,thereareseveralbiotransformationreactions
thatexclusivelyandpredominantlytakeplaceinthe‘buffer
solution’andthiseventuallyrenderstheultimaterecoveryof
the‘desiredproduct’relativelyeasyandconvenient.
22

23. 3.ImmobilizedCells
•Inmorerecenttimes,ahostofbiotransformation
methodologiesdomakeuseoftheimmobilizedcellsthus
affordingthebiggestevenadvantageouspluspointthatthe
processcouldbecarriedoutsimultaneously;besides,thecells
mightbeemployedoverandoveragain.
Applications:Inactualpractice,theimmobilizedbacterial
cellsthatinvariablycatalyzeeithersingle-stagereactionor
multi-stagereaction,arepresentlyexploitedinthelarge-scale
productionofL-alanine,asparticacid,andmalicacid.
23

24. Selection of organism
•Theselectionofstrainseitherfromitsnaturalsourcesor
fromthevariousavailablecultureswhicharesolely
responsibleforcatalyzingthedesiredbiotransformation
reaction(s)isnotonlyvitalandcriticalbutalsoofgreat
importance.
•Ithasbeenobservedthattherearequiteafew
microorganismsthatusuallycarryoutthedesired
bioconversionswiththehelpofarelatedchemicalentity.
•Insteroidonemayencounteraratherdifficultproblemdueto
thelackofselectivemethodssoastoidentifythecolonies
preciselywhichusuallyperformtheappropriatespecific
activity.
24

25. 1.ModifiedEnrichmentMethod:
•Themodifiedenrichmentmethodisinvariablyusedfortheisolationof
mutantsblockedinthesubstratedissimilationmechanism.
•Inthisspecificinstance,asteroidsubstrateisnormallyincorporatedasthe
soleC-sourceexclusivelyina‘minimalmedium’seededadequatelywith
thesoildilutions.
•Thecellsthatcausesthedegradationofthesubstratewillultimatelygrow
;andare,therefore,subsequentlytransferredtothesamemediumbut
particularlyenrichedwithanotherC-source,forinstance:glucose.
•However,themutantsmaybepresentwhicharestrategicallyblockedat
differentstagesintheprocessofdegradationofthesteroidsubstrate,but
mayconsumeglucoseastheC-source.
•Ithasbeenprofuselyestablishedandreportedthatafairlylargenumber
ofmicrobialstrainsviz.,eubacteria,yeasts,molds,andstreptomycetes
maybestoredandmaintainedstrictlyaspertherecommended‘standard
methods’,suchas:agarslant,soilculture,frozenculture,andlypholized
culturepreservedattemperaturesrangingbetween–20°Cto–170°C.
25

26. •Besides,theresultingintermediatesmaygetaccumulated,
whereasthelesion-bearingmutantscanbeisolated
conveniently.Furthermore,mutantsmayalsobeisolated
whichareincapableofaccumulatingan‘undesirable
compound’.
26

27. 2.FiltrationEnrichmentMethod:
•Inthiscase,aftermutagenesisthesporesoffilamentous
organismse.g.,actinomycetes,fungi,aremadetodevelopina
liquidminimalmedium.
•Themicrocoloniesofprototrophsthusdevelopedare
meticulouslyseparatedbyfiltration,wherebythesporesof
auxotrophsthatwereunabletogrowleftbehindinthe
filtrate.
•Thefiltrateobtainedinthismannerinsubsequentlyplated
andtheresultingcoloniesareadequatelycheckedfor
auxotrophiccharacteristics.
27

28. 3.Penicillin-SelectionProcedure:
•Inpenicillin-selectionproceduretheprevailinggrowingcellsarekilled
selectivelybythe‘antibiotic’treatment,therebyenrichingtheauxotrophs
thatareincapableofgrowinguponthe‘minimalmedium’.
•Thus,exclusivelybasedupontheirmodeofactionaplethoraof‘inhibitors’
otherthanpenicillinmayalsobeemployedeffectivelyinthisprocedure,
namelydihydrostreptomycinforPseudomonasaeruginosa;nystalinfor
Hansenulapolymorpha,Penicilliumchrysogenum,Aspergillusnidulans,
andSaccharomycescerevisiae;nalidixacidforSalmonellatyphimurium;
colistinforthepenicillin-resistantHydrogenomonasstrainH16.
4.SodiumPentachlorophenolate:Thesaltsodiumpentachlorophenolate
alsoaffordsenrichmentprocedurebyvirtueofitsgreatertoxicityparticularly
againstthe‘germinatingspores’incomparisontothe‘vegetativecells’.
Example:Theabovemethodhasbeensuccessfullyappliedwithseveral
organisms,suchas:Penicilliumchrysogenum;Streptomycesaureofaciens;
Streptomycesolivaceus;andBacillussubtilis.
28

29. 5.SprayingwithReagents(orIncorporatingIndicatorDyes):
Onemayobserveeitherthepresenceorabsenceofspecificenzymeactivities
almostdirectlyinthecoloniesthatareallowedtogrownonplatesby
employingeitherofthetwoavailablecommonprocedures,namely:(a)
sprayingwithappropriatereagents;and(b)incorporatingindicator-dyes
rightintotheculturemedium.
6.InhibitionofAssayOrganisms:Inthisspecificinstancetheantibiotically-
activecompoundsmaybedetectedquiteeasilyandconvenientlyby
measuringtheinhibitionofsensitiveassayorganisms.Thisprocedureallows
theprecisedetermination(assay)ofthe‘antibioticcontent’ofanunknown
solutionusingareferencestandardsimultaneously.
7.AgarPlugMethod:Theagarplugmethodisregardedtobeoneofthe
mostreliableandprecisetechniqueswhereintheagarcylindershaving
‘single-colonies’aretransferredtotestplatesafterdueincubationpreferably
inamoistchamberasdepictedinfiguregivenbelow:
29

30. 30