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.
7 WorksCited
Curylo, Elisabeth A., et al. Isolation and Antimicrobial Potential of Epsilon Poly-L-Lysine. Ed. Jill
Rulfs, Theodore C. Crusberg, and Michael Allan Buckholt. Vol. E-project-042408-104838.
pg.20
Worcester Polytechnic Institute:, 2008. <http://www.wpi.edu/Pubs/E-project/Available/E-
project-042408-104838/>.
Geornaras, Ifigenia, and John N. Sofos. "Activity of ε–Polylysine Against Escherichia Coli
O157:H7, Salmonella Typhimurium, and Listeria Monocytogenes." Journal of Food
Science 70.9 (2005): M404-8. <http://dx.doi.org/10.1111/j.1365-
2621.2005.tb08325.x>.
Hamano, Y., et al. "Ɛ-Poly-l -Lysine Producer, Streptomyces Albulus, has Feedback-Inhibition
Resistant Aspartokinase." Applied Microbiology and Biotechnology 76.4 (2007): 873-82.
<http://dx.doi.org/10.1007/s00253-007-1052-3>.
Hamano, Yoshimitsu, et al. "Development of Gene Delivery Systems for the ɛ-Poly-L-Lysine
Producer, Streptomyces Albulus." Journal of Bioscience and Bioengineering, 99.6 (2005):
636-41. .
Hiraki, J. "Use of ADME Studies to Confirm the Safety of Epsilon-Polylysine as a Preservative in
Food." Regulatory Toxicology and Pharmacology : RTP 37.2 (2003): 328. . EBSCO:.
Hirohara, Hideo, et al. "Substantially Monodispersed Poly(ɛ-l -Lysine)s Frequently Occurred in
Newly Isolated Strains of Streptomyces Sp." Applied Microbiology and Biotechnology 76.5
(2007): 1009-16. <http://dx.doi.org/10.1007/s00253-007-1082-x>.
Kahar, Prihardi, et al. "Enhancement of ɛ-Polylysine Production by Streptomyces Albulus Strain
410 using pH Control." Journal of Bioscience and Bioengineering, 91.2 (2001): 190-4. .
Kahar, Prihardi, et al. "Production of ɛ-Polylysine in an Airlift Bioreactor (ABR)." Journal of
Bioscience and Bioengineering, 93.3 (2002): 274-80. .
Kito, Mitsuaki, et al. "Purification and Characterization of an ɛ-Poly--Lysine-Degrading Enzyme
from the ɛ-Poly--Lysine-Tolerant Chryseobacterium Sp. OJ7." Journal of Bioscience and
Bioengineering, 96.1 (2003): 92-4. .
pg.21
Kunioka, M. "Biosynthesis and Chemical Reactions of Poly(Amino Acid)s from Microorganisms."
Applied Microbiology and Biotechnology 47.5 (1997): 469-75.
<http://dx.doi.org/10.1007/s002530050958>.
Malachowski, Lisa Lyn. Investigation of Immobilized Biopolymers for Metal Binding [Electronic
Resource].
http://www.lib.utexas.edu/etd/d/2004/malachowskil62902/malachowskil62902.pdf
University of Texas, 2006 9/15/2008.
Nishikawa, Masanobu, and Ken'ichi Ogawa. "Inhibition of Epsilon-Poly-L-Lysine Biosynthesis in
Streptomycetaceae Bacteria by Short-Chain Polyols." Applied and Environmental
Microbiology 72.4 (2006): 2306-12. .
Rao Popuri, S., et al. "Biosorption of Hexavalent Chromium using Tamarind (Tamarindus
Indica) Fruit Shell - A Comparative Study." Electronic Journal of Biotechnology Vol. 10,
No. 3.July 15, 2007 (2007): 9/14/2008. . 9/14/2008.
Shih, Ing-Lung, Ming-Haw Shen, and Yi-Tsong Van. "Microbial Synthesis of Poly(ε-Lysine) and
its various Applications." Bioresource Technology, 97.9 (2006): 1148-59. .
Shima, S., and H. Sakai. "Poly-L-Lysine Produced by Streptomyces. Part III. Chemical
Studies." Agricultural and Biological Chemistry 45.11 (1981): 2503. . EBSCO:.
---. "Polylysine Produced by Streptomyces. Argic." The Journal of Biological Chemistry 41
(1977): 1807. . EBSCO:.
Shirling, Elwood B., and David Gottlieb. "Cooperative Description of Type Strains of
Streptomyces: V. Additional Descriptions." International Journal of Systematic and
Evolutionary Biology 22.4 (1972): 265-394. .
Stokes, J. L., and Marion Gunness. "The Amino Acid Composition of Microorganisms." The
Journal of Bacteriology 52.2 (1946): 195-207. .
pg.22
Szókán, Gy, et al. "Structure Determination and Synthesis of Lysine Isopeptides Influencing on
Cell Proliferation." Biopolymers 42.3 (1997): 305-18.
<http://dx.doi.org/10.1002/(SICI)1097-0282(199709)42:3<305::AID-BIP4>3.0.CO;2-
Q>.
Tarantino, Laura. "FDA/CFSAN: Agency Response Letter: GRAS Notice No. GRN 000135."
Office of Food Additive Safety. January 16, 2004 2004. 12/11/2008
<http://www.cfsan.fda.gov/~rdb/opa-g135.html>.
Yoshida, T., and T. Nagasawa. "Imicro-Poly-l-Lysine: Microbial Production, Biodegradation and
Application Potential." Applied Microbiology and Biotechnology 62.1 (2003): 21. .
EBSCO:.
