Epsilon‐Poly‐L‐LysineProducedbyS.albulusAMajorQualifyingProjectReport

SubmittedtotheFacultyOfthe

WORCESTERPOLYTECHNICINSTITUTEInpartialfulfillmentoftherequirementsforthe

DegreeofBachelorofScienceby

MaureenE.Ryder

2010

Approved:

_________________________________________MichaelA.Buckholt

_________________________________________AllisonHunter

pg.2

ContentsAbstract.......................................................................................................................................................... 31 Whatisε‐Poly‐L‐Lysine?................................................................................................................. 4

1.1 Usesofε‐Poly‐L‐Lysine........................................................................................................... 41.2 StructureandPropertiesofε‐Poly‐L‐Lysine ................................................................. 6

2 BacteriaUsed ....................................................................................................................................... 72.1 Streptomycesalbulus ................................................................................................................ 72.2 Pediococcusacidilactici............................................................................................................ 8

3 Goals ........................................................................................................................................................ 84 MethodsandMaterials .................................................................................................................... 94.1 ReconstitutionandCreationofFreezerStock .............................................................. 94.2 ThinLayerChromatography ................................................................................................ 94.3 DeterminationofLysinelevelsinS.albuluscells ......................................................11

5 Results ..................................................................................................................................................145.1 ReconstitutionandCreationofFreezerStock ............................................................145.2 ThinLayerChromatography ..............................................................................................145.3 DeterminationofLysinelevelsinS.albuluscells ......................................................14

6 Discussion ...........................................................................................................................................177 WorksCited ........................................................................................................................................19

pg.3

Abstract

Sinceitsdiscoveryin1977,ε‐Poly‐L‐Lysine(ε‐PL)hasbeenusedinavariety

ofapplications.Theprimaryusageofε‐PLcurrentlyisasananti‐microbialfood

additive,thoughitcanbefoundasadrugdeliverycarrier,hydrogel,endotoxin

remover,biosensor,ordisinfectant,amongotherpossibilities.Thegoalofthis

projectwasprimarilytoinvestigatewhetheraparticularstrainofStreptomyces

albulusthatallegedlyproducesε‐Poly‐L‐Lysinecouldbeusedasafuturepositive

controlforε‐Poly‐L‐Lysineproduction.

pg.4

1 Whatisε­Poly­L­Lysine?

1.1 Usesofε­Poly­L­Lysine

Table1.PotentialApplicationsofEpsilonPolylysineanditsDerivatives.(Shih2006)

Sinceitsdiscoveryin1977,ε‐Poly‐L‐Lysinehasbeenusedforavarietyof

applications.Theprimaryusageofε‐PLcurrentlyisasananti‐microbialfood

additive,thoughitcanbefoundasadrugdeliverycarrier,hydrogel,endotoxin

remover,biosensor,ordisinfectant,amongotherpossibilities.Manyofthe

applicationsareparticularforε‐PLanditsderivativesasopposedtoα‐PLduetoa

chemicalmodificationonthealphaaminogroup,whichcanhinderitsadsorption

(Yoshida2003).

Theproposedmechanismfortheantimicrobialactivityofε‐PLinvolvesthe

molecules’adsorptionontoacellsurface,leadingtoastrippingoftheouter

membraneandconsequentabnormaldistributionofcytoplasm,bringingaboutcell

pg.5

death.(Geornaras2005,Yoshida2003).Thismechanismcanalsoexplainthe

differencesthathavebeenobservedinantimicrobialeffectivenessonvarious

organisms;differencesincellsurfaceswouldleadtodifferencesinadsorptionlevels

(Yoshida2003).ε‐Poly‐L‐Lysinecaninhibitgrowthinavarietyoforganismsat

concentrationsoflessthan100µg/mL,thoughtheamountneededforfood

preservationislessthan300ppm(Yoshida2003).Theanti‐microbialactivityofε‐PL

hasleadtoitsuseasafoodpreservativeinJapansincethe1980s,infoodssuchas

boiledriceandslicedfish(Hiraki2003)andin2004ε‐PLwasdeemedGenerally

RecognizedAsSafe(GRAS)bytheUSFoodandDrugAdministrationforuseasa

foodpreservativeinamountslessthan50mg/kg(Tarantino2004).

ε‐Poly‐L‐Lysineisnotonlyusefulasanantimicrobialagent;ithaspotentialto

beanefficient,environmentallyfriendlybioremediator.Itscationicpropertiesbring

upthepotentialabilityforheavymetalbinding.Additionally,itisabiodegradable

non‐toxicpolymer,whichmeansitwouldbesafefortheenvironment.Duetoits

cationicnature,itishypothesizedthatεPLmayhavetheabilitytoreducechromate

andotheranionicheavymetalwastesfromindustrialdischargethrough

biosorption.Biosorptionisapossiblycosteffectivewayofremovingheavymetals

fromindustrialwastewaters(RaoPopuri2007).Thishasnotbeensufficiently

explored,thoughifε‐Poly‐L‐Lysinewereabletoremoveheavymetalsfromwater

sourcesitwouldbeaninvaluableresource.

pg.6

1.2 StructureandPropertiesofε­Poly­L­Lysine

Figure1.Structureofε­Poly­L­Lysine(Hirohara2007)

ε‐Poly‐L‐Lysine(ε‐PL)isahomo‐polymericcompoundof25‐30lysine

monomerslinkedattheε‐aminogroups(Shima1981).Itisabiodegradable,non‐

toxic,andwatersolublesubstancesecretedinvaryingamountsbythefilamentous

bacteriaStreptomycetaceae(Shih2006,Shima1981).ε‐PLinwatercontainsa

positivelychargedhydrophilicaminogroupandahydrophobicmethylenegroup

thatleadsittohaveinterestingandusefulcationicproperties,discussedlaterinthis

section.ε‐PLisunlikeproteinsandα‐poly‐L‐Lysine(α‐PL)inthattheamidelinkage

inε‐PLisbetweentheε‐aminocarbonandthecarbonylgroup(seeFigure1)while

theamidelinkageinα‐PLisfoundbetweentheα‐aminocarbonandthecarboxyl

group(Shima1981).TheaminoacidLysinehasapositivelychargedε‐aminogroup,

whichcausesapolymeroflysineresiduestobehighlypositivelycharged.Ina

hydrophilicenvironment,thecarboxylandaminogroupsarrangethemselves

towardstheoutsideofaglobularstructure,whilethehydrophobicmethylene

groupsarearrangedtowardstheinsideoftheglobularstructure.

In1977,ShimaandSakaiwerethefirsttodiscoverε‐PLasaresultof

screeningforDragendorff’spositivesubstancesproducedbyStreptomycesalbulus

pg.7

ssp.Lysinopolymerusstrain346(Shima1997).Dragendorff’sreagentactivatesinthe

presenceofalkaloidsubstances,alertingtheresearchersthatamoleculeofinterest

waspresent.Becauseofthis,manyearlypapersrefertoε‐PLastheDP

(Dragendorff’sPositive)substance,beforethestructurewasdeterminedanditwas

properlynamed.Conventionalindustrialpolypeptideandpoly‐aminoacid

synthesisregimescannotproperlyformthenecessaryεbonds,leadingtothe

practiceofε‐Poly‐L‐Lysinebeingproducedthroughfermentationwithbacteriaand

subsequentlyextracted.PrihardiKaharhasestablishedaprotocolfortheefficient

fermentationofStreptomycesalbulusstrain410usingpHcontrol,andhas

subsequentlyrefinedherdatathroughinvestigationofproductionofε‐PLinand

AirliftBioreactor(Kahar2001,2002).

2 BacteriaUsed

2.1 Streptomycesalbulus

Theprimarysourceforε‐Poly‐L‐Lysine

acrossavailableliteratureprovestobe

Streptomycesalbulus(Hirohara2007).

Picturedatleft,S.albus,acloselyrelated

species,canbeseenshowingan

undulatemarginina10‐daycultureon

nutrientagar.ThegenusStreptomycesis

ofthephylumActinobacteria,which

Figure2.IrregularformofStreptomycesalbus,acloserelativeofStreptomycesalbulus(BryanMacDonald,ChristopherAdams,andKyleSmith,BrighamYoungUniversity,Provo,UT)

pg.8

impliesthatS.albulusisgrampositive.Streptomycesbacteriaaretypicallyfound

insoilordecayingvegetation,andhaveacharacteristicallycomplexsecondary

metabolism,whichmakesthemaninvaluableresourceforpharmaceutical

manipulationfordrugproduction(Shirling1972).

ThestrainofStreptomycesalbulususedinthisprojectisaknownproducerof

ε‐Poly‐L‐Lysine.Itisslowergrowingthanalotofcommonlaboratorybacteria,

oftenneeding3‐5daysincubationforsufficientgrowth.

2.2 Pediococcusacidilactici

Pediococcusbacteriaaregram‐positivebacteriathatgenerallyappearin

pairsortriads.ThisspeciesisconsideredaLacticAcidBacteria,soitcan

toleratelowerpHranges,isnon‐respirating,andcreateslacticacidasthe

metabolicendproductofcarbohydratefermentation.Theparticularstrain

usedinthisprojectisL‐Lysinesensitive;itcannotsynthesizelysinede­novo,

andmusttakeitupfromthesurroundingenvironment.

3 Goals

Thegoalsofthisprojectare:

1.) Toobtainbacteriaabletoproduceε‐Poly‐L‐Lysinefroman

alternateresearchestablishmentorresearchsupplysource.

pg.9

2.) Tofindanadequatemethodofdetectionofpresenceofε‐Poly‐L‐

lysine(positivecontrolneeded)andusethistodetermine

presenceofε‐Poly‐L‐lysineproducedbybacteria.

3.) Hypothesis:Iftheobtainedbacterialstrainproducesε‐Poly‐L‐

lysine,thenasignificantabundanceoflysinemonomerswillbe

showntobepresentinthestrainofbacteria.

4 MethodsandMaterials

Therewerevarioustechniquesusedinthisprojecttotryanddeterminethe

presenceofε‐Poly‐L‐lysineandproveitsabundanceinS.albulus.

4.1 ReconstitutionandCreationofFreezerStock

ThevialcontainingS.albuluswasheatedandcrackedand5.0mLofsterilized

LBbrothwasaddedtoreconstitutethedriedbacteria.Thismixturewas

transferredto5.0mLLBbrothandM3Gmediainconicaltubes,andallowedto

incubatefor5days.After5days,3vialsof1.0mLLBincubatedS.albuluswere

addedto40%glycerolinH2Otoacryogenicvialandplacedintothe‐80oC

freezer.

4.2 ThinLayerChromatography

ThinLayerChromatographywasusedtoseparatesolutionsofdifferenttypes

oflysine,todetermineifthesewouldtraveldifferentlyandtherebybeableto

pg.10

bedifferentiatedfromeachother.Thesolutionusedtoseparatethe

compoundswasa4:1:1:2ratioofn‐butanol:glacialaceticacid(17.4M):

pyridine:dH2O.Theε‐Poly‐L‐lysinethatwasordereddidnoteasilydissolvein

eitherH2Oorethylalcoholasoriginallyexpected.Uponfurtherinvestigation

andcontactingthesupplier,itwasdiscoveredthatthesidechainswere

protectedusingCarboxybenzyl,whichbegsthequestionofitsusefulnessifthe

sidechainsareunabletointeractwithstationaryormobilephaseoftheTLC.

Theε‐Poly‐L‐lysinewasfinallyfoundtodissolveindichloromethane.

Thesilicagelplateswerespottedwiththefollowinglanes:

Table2.LaneDesignationforSilicaGelChromatography

Plate1

Lane Compound Volume

1 α‐Poly‐L‐lysine 2µL

2 α‐Poly‐L‐lysine 20µL

3 ε‐Poly‐L‐lysine 2µL

4 ε‐Poly‐L‐lysine 20µL

5 S.albulusinLBbroth 10µL

6 S.albulusinLBbroth 20µL

7 S.albulusinM3Gmedia 10µL

Plate2

Lane Compound Volume

1 S.albulusinLBbroth 20µL

2 LBbroth 20µL

3 S.albulusinM3Gmedia 20µL

4 M3Gmedia 20µL

pg.11

AfterplacingtheplateinaTLCtankandallowingthechromatographytorun

forabout4hours,theplatewasremovedfromthemobilesolutionand

allowedtodry.Upondrying,theplatewassprayedwithaninhydrinsolution

(0.5mLninhydrinstock+15mLethanol),whichwasusedtovisualizethe

presenceofaminoacids.

4.3 DeterminationofLysinelevelsinS.albuluscells

Thetestorganism,Pediococcusacidilacticiwasreconstitutedandsubsequently

subculturedinto10mLDifcoMicroAssayCultureBroth(inoculumbroth)and

incubatedat37°Cfor24hours.Theresultantsuspensionwascentrifugedat

600gfor3minutestoseparatecellpelletfromtheliquidsupernatant.The

supernatantwasremovedandcellswerere‐suspendedin10mLof0.85%

(w/v)sterilesalinesolutiontowashthem,andthencentrifugedandwashed

twomoretimesin10mLof0.85%(w/v)sterilesalinesolutiontoremove

lingeringinoculumbroth.1.0mLofresuspendedcellsolutioninsalinewas

dilutedwith19.0mL0.85%(w/v)sterilesalinesolutiontocreatethediluted

inoculumsuspension.

ThestocksolutionofL‐lysinemonomerswaspreparedfreshtoprevent

degradation.0.001gL‐Lysinewasdissolvedinto20.0mLpurifiedH2Oto

createtheL‐Lysinestocksolution.0.5mLofStockSolutionwasdilutedwith

416mLpurifiedH2OtocreatetheStandardWorkingSolutionwithalysine

concentrationof6.0mg/mL.Ninetubeswereusedforthestandardcurve

pg.12

determination.Eachtubecontained5.0mLAssaybrothwiththefollowing

concentrationsofpurifiedwaterandStandardWorkingSolution:

Table3.StandardCurveSolutions

Tube Standard

WorkingSolution

(mL/10mLtube)

PurifiedH2O

(mL/10mL

tube)

FinalAmino

Acid

Concentration

(mg/mL)

Blank 0.0 5.0 0

2 0.5 4.5 3

3 1.0 4.0 6

4 1.5 3.5 9

5 2.0 3.0 12

6 2.5 2.5 15

7 3.0 2.0 18

8 4.0 1.0 24

9 5.0 0.0 30

Eachtubewasinoculatedwith100µLDilutedInoculumSuspensionand

allowedtoincubatefor20hoursat37C.Growthresponsewasmeasured

turbidometricallyviaspectrophotometryat660nm.

Theexperimentalorganism,S.albuluswassubculturedininoculumbrothat

37Cforfivedays.Similartothetestorganism,thesecellswerecentrifuged

andwashedin10mLof0.85%(w/v)sterilesalinesolutionthreetimes.The

cellularpelletwasremovedandweighedbysubtractingtheweightofasterile

conicaltubefromtheweightofthetubewiththepellet.0.02gofcellular

pelletwaslysedusingamechanical‐blendingtechniqueofplacingasterile

pg.13

toothpickintothetubeandvortexingfor2minutes.Thislysatesuspension

wasdilutedtoatotalvolumeof20mL.TheDilutedCellularLysatewasadded

inincreasingamountstoaseriesoftubeseachcontaining5.0mLofAssay

mediumaswellasthefollowingconcentrationsofpurifiedwater:

Table4.CellularLysateSolutions

Tube

DilutedCellular

Lysate(mL/10mL

tube)

PurifiedH2O

(mL/10mLtube)

Blank 0 5.0

2 0.5 4.5

3 1 4.0

4 1.5 3.5

5 2 3.0

6 2.5 2.5

7 3 2.0

8 4 1.0

9 5 0.0

Eachtubewasinoculatedwith100µLDilutedInoculumSuspensionand

allowedtoincubatefor20hoursat37C.Growthresponsewasmeasured

turbidometricallyviaspectrophotometryat660nm.

pg.14

5 Results

5.1 ReconstitutionandCreationofFreezerStock

TheS.albulusobtainedwassuccessfullygrowninLBbrothandM3Gminimal

media.Thefreezerstocksweresuccessfullyreconstitutedthusshowingthe

effectivelong‐termstorageofthe‐80oCfreezer.

5.2 ThinLayerChromatography

TheTLCplateswereinconclusive,asaminoacidsinthebacteriaandgrowth

mediacausedallofthespotsfromS.albulustoshowapositiveresultinthe

ninhydrinstain.

5.3 DeterminationofLysinelevelsinS.albuluscells

Asexpected,theabsorbanceat660nmincreasedalongwiththeincreasing

amountoflysinemonomersinsolution.Atlowlevelsoflysinenoabsorbance

wasseen;thisimpliesthatiftherewasanyexperimentalorganismgrowthin

thesetubes,thelevelsweretoolowtobecalculatedbythe

spectrophotometer.Thesedatapointswereleftoutofthestandardcurve

trendlinecalculation,astheydonotaccuratelyreflectgrowth.Asseenin

Figure3,theStandardCurvedeterminedbytheknownlevelsoflysine

solutionresultedinthecalculatedtrendlineshowingthatAbsorbance=

0.0006(Lysine[µg/mL])–0.0053.Thisinformationwaslatercomparedto

unknownlevelsoflysinesolutionstodeterminetheaveragelysinelevelper

mL.

pg.15

Table5.StandardcurvedatawithknownconcentrationsofLysine.

Tube Standard Working Solution (mL/10mL tube)

Purified H2O (mL/10mL tube)

Final Amino Acid Concentration (µg/mL)

%T at 660nm Absorbance at 660nm

1 0.0 5.0 0 100 0 2 0.5 4.5 3 100 0 3 1.0 4.0 6 100 0 4 1.5 3.5 9 100 0 5 2.0 3.0 12 99.6 0.001740662 6 2.5 2.5 15 99.4 0.002613616 7 3.0 2.0 18 98 0.008773924 8 4.0 1.0 24 97.4 0.011441043 9 5.0 0.0 30 97.3 0.01188716

Figure3.LysineStandardCurve(blue)withcalculatedtrendline(black)

Table6.Absorbancedatafortestorganisminunknownlysinelevelsfromcellularlysate.

TubeDilutedCellularLysate(mL/10mLtube)

PurifiedH2O(mL/10mLtube)

%Tat660nm

Absorbanceat660nm

1 0 5.0 100 0

pg.16

2 0.5 4.5 100 03 1 4.0 99.8 0.0008694594 1.5 3.5 99.8 0.0008694595 2 3.0 99.3 0.0030507526 2.5 2.5 98.4 0.0070049027 3 2.0 98.5 0.006563778 4 1.0 98.5 0.006563779 5 0.0 97.7 0.010105436

Figure4.GrowthofTestOrganism

TheadditionofdilutedS.albuluscellularlysateinincreasingamountsallows

forthecalculationoftheconcentrationoflysineinthecellsviacomparisonto

theStandardCurveseeninFigure3.ThedatainFigure4wasusedto

determinethemeanconcentrationoflysinefoundinthedilutedcellular

pg.17

lysate,5.66µg/mL.Recallingthat0.002gofcellularlysatewasoriginally

dilutedto20mL,thisgivesthetotalcelllysate/lysineratiotobe17.668by

mass.

6 Discussion

Thehypothesisofthisprojectstatesthatiftheobtainedbacterialstrain

producedε‐Poly‐L‐lysine,thenasignificantabundanceoflysinemonomers

wouldbeshowntobepresentinthestrainofbacteria.S.albuluswasshownto

containhighlevelsoflysine;about1/18ofthecells’masswaslysine.This

wouldbeconsistentwithanorganismthatproducesapoly‐lysine;an

overabundanceoflysinewouldbenecessaryfortheproductionofalysine

polymer.Anon‐poly‐lysineproducingstrainofStreptomycesgriseuscontained

anaverageof2.13%lysinepercellbyweight(Stokes,1946),whilethe

experimentalstraininthisprojectshowedanaverage5.55%lysinepercellby

weight.Thisdifferenceisstatisticallysignificant(p=0.00159)bytheStudent’s

T‐test(p<0.05showssignificance.)However,thisexperimentwasonly

performedonce,sorepeatedresultswouldgivemoreweighttothese

conclusions.

TheresultsreceivedintheTLCexperimentwereabletodeterminethatthere

wereaminoacidsinthegrowthmediaandcells,butduetothenon‐specificity

oftheninhydrinstainthepresenceoflysinecouldnotbeseparatedfromthe

pg.18

presenceofanyotheraminoacid.Thesameoutcomewouldhavebeenobserved

ifonlythecellpelletshadbeenrunontheTLC,ascellscontainmanyamino

acidsandproteins.Ninhydrinisaqualitativeindicator,notquantitative,and

therebyitisinadequatetoshowanoverabundanceoflysineinS.albuluscells.

Thisexperimentwouldhavebeenmoresuccessfulifalysinespecificstaincould

befound.Anegativelychargedstainthatwouldbeabletobindtopositively

chargedmoleculescouldworklimitedly;poly‐lysinewouldnotbetheonly

positivelychargedmoleculesinacell,sothismethodwouldstillnotbespecific

enoughtodefinitivelydeterminepoly‐lysineproduction.

Eventhoughε‐Poly‐L‐LysineissecretedintotheenvironmentbyS.albulus,the

cellpelletwasusedinthisexperimentbecausetheobjectivewastoshowan

abundanceoflysinemonomers,nottheε‐Poly‐L‐Lysinemoleculeitself.Ifone

weretobelookingfororidentifyingε‐Poly‐L‐LysinefromS.albulus,thegrowth

mediashouldbeseparatedfromthebacterialpelletafterfermentationandthe

growthmediashouldbeused.Theexperimenttestinglysinelevelscannot

determinethetypeofpoly‐lysinethatmightbeproducedbythebacteria.IfS.

albulusproducedα‐poly‐L‐lysineasopposedtoε‐poly‐L‐lysine,thesesame

resultswouldstillbeexpected.Becausethisdistinctionhasnotbeen

determinedinthisproject,thisstraincannotdefinitivelybedeclaredapositive

ε‐Poly‐L‐Lysineproducer,thoughevidenceseemstopointthatway.Further

investigationwouldbenecessarytodeterminewhichtypeofpoly‐L‐lysineis

produced.

pg.19

Additionally,highlevelsofε‐Poly‐L‐Lysinewouldhaveaquenchingeffecton

experimentalbacterialgrowth.Inordertopreventthisfromhappening,one

wouldhavetohydrolyzetheproteins(and,intheprocess,anypoly‐lysine

molecules).Doingthiswouldstillbeabletoshowheightenedlevelsoflysine

comparedtonon‐producerswithouttheriskofquenchingskewingtheresults.

Sinceε‐Poly‐L‐Lysinehasbeenshowntobebiodegradableandnontoxic

towardshumansandtheenvironment,(Shih,Shen,&Van,2006)ε‐Poly‐L‐

Lysineanditsderivativeshavebeenstudiedforawiderangeofapplications

includingfoodpreservation,dietaryagent,creationofhydrogels,andmore.

Furtherstudyofε‐Poly‐L‐Lysineproducerscanhelpstreamlinetheproduction

processandbringdowncosts.Duetoitscationicnature,itishypothesizedthat

εPLmayhavetheabilitytoreducechromateandotheranionicheavymetal

wastesfromindustrialdischargethroughbiosorption.Biosorptionisa

potentiallycosteffectivewayofremovingheavymetalsfromindustrial

wastewaters(RaoPopuri,Jammala,Reddy,&Venkata,).Ifanadequate

producerofε‐Poly‐L‐Lysinewerefound,thisapplicationwouldprovidean

environmentallysoundbiosorptiontechnique.

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