Amino-Acid Homopolymers Occurring in Nature PDF
Preview Amino-Acid Homopolymers Occurring in Nature
Microbiology Monographs Volume 15 Series Editor: Alexander Steinbu¨chel Mu¨nster,Germany Microbiology Monographs Volumes published in the series InclusionsinProkaryotes HydrogenosomesandMitosomes: VolumeEditor:JessupM.Shively MitochondriaofAnaerobicEukaryotes Vol.1,2006 VolumeEditor:JanTachezy Vol.9,2008 ComplexIntracellularStructures inProkaryotes UncultivatedMicroorganisms VolumeEditor:JessupM.Shively VolumeEditor:SlavaS.Epstein Vol.2,2006 Vol.10,2009 MagnetoreceptionandMagnetosomes MicrobialMegaplasmids inBacteria VolumeEditor:EdwardSchwartz VolumeEditor:DirkSchu¨ler Vol.11,2009 Vol.3,2007 EndosymbiontsinParamecium PredatoryProkaryotes–Biology,Ecology VolumeEditor:MasahiroFujishima andEvolution Vol.12,2009 VolumeEditor:EdouardJurkevitch Vol.4,2007 Alginates:BiologyandApplications VolumeEditor:BerndH.A.Rehm AminoAcidBiosynthesis–Pathways, Vol.13,2009 RegulationandMetabolicEngineering VolumeEditor:VolkerF.Wendisch Vol.5,2007 PlasticsfromBacteria:NaturalFunctions andApplications VolumeEditor:Guo-QiangChen MolecularMicrobiologyofHeavyMetals Vol.14,2010 VolumeEditors:DietrichH.Nies andSimonSilver Vol.6,2007 Amino-AcidHomopolymersOccurring inNature VolumeEditor:YoshimitsuHamano MicrobialLinearPlasmids Vol.15,2010 VolumeEditors:FriedhelmMeinhardt andRolandKlassen Vol.7,2007 ProkaryoticSymbiontsinPlants VolumeEditor:KatharinaPawlowski Vol.8,2009 Yoshimitsu Hamano Editor Amino-Acid Homopolymers Occurring in Nature Editor YoshimitsuHamano,Ph.D. DepartmentofBioscience FukuiPrefecturalUniversity 4-1-1Matsuoka-Kenjojima,Eiheiji-cho Fukui910-1195 Japan [email protected] SeriesEditor ProfessorDr.AlexanderSteinbu¨chel Institutfu¨rMolekulareMikrobiologieundBiotechnology Westfa¨lischeWilhelms-Universita¨t Corrensstr.3 48149Mu¨nster Germany [email protected] ISSN1862-5576 e-ISSN1862-5584 ISBN978-3-642-12452-5 e-ISBN978-3-642-12453-2 DOI10.1007/978-3-642-12453-2 SpringerHeidelbergDordrechtLondonNewYork LibraryofCongressControlNumber:2010930857 #Springer-VerlagBerlinHeidelberg2010 Thisworkissubjecttocopyright.Allrightsarereserved,whetherthewholeorpartofthematerialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting, reproductiononmicrofilmorinanyotherway,andstorageindatabanks.Duplicationofthispublication orpartsthereofispermittedonlyundertheprovisionsoftheGermanCopyrightLawofSeptember9, 1965,initscurrentversion,andpermissionforusemustalwaysbeobtainedfromSpringer.Violations areliabletoprosecutionundertheGermanCopyrightLaw. Theuseofgeneraldescriptivenames,registerednames,trademarks,etc.inthispublicationdoesnot imply, even in the absence of a specific statement, that such names are exempt from the relevant protectivelawsandregulationsandthereforefreeforgeneraluse. Coverdesign:SPiPublisherServices Printedonacid-freepaper SpringerispartofSpringerScience+BusinessMedia(www.springer.com) Preface Biopolymersarethemostabundantmoleculesinlivingmatter.Microorganismsare capable of producing a wide variety of biopolymers, including polynucleotides, polyamides(protein),polysaccharides,polyphosphate,polyesters,andpolyketides. However,homopolymers,whicharemadeupofonlyasingletypeofaminoacid, are far less ubiquitous; in fact, only two amino-acid homopolymers are known to occur in nature: poly-e-L-lysine (e-poly-L-lysine, e-PL) and g-poly-glutamic acid (g-PGA). e-PL,consistingof25–30L-lysineresidueswithalinkagebetweenthea-carboxyl group and the e-amino group, is produced by actinomycetes. Because e-PL is a polycationicpeptideandthusexhibitsantimicrobialactivityagainstawidespectrum of microorganisms, including Gram-positive and Gram-negative bacteria, and because it is both safe and biodegradable, e-PL is used as a food preservative in several countries. In contrast, g-PGA is an unusual anionic polypeptide in which D-and/orL-glutamateispolymerizedviag-amidelinkages.g-PGAissecretedintothe growthmediumofBacillussubtilisasafermentationproductwithavariablemolecu- larweight(typically,10–1,000kDa). Overthepastdecade,thebiologicalandchemicalfunctionsofthesetwohomo- polyaminoacidshavebeenreported,therebybeingpromisingmaterialsformedi- calandindustrialapplications.ThisMicrobiologyMonographsvolumecoversthe current knowledge and most recent advances in regard to the occurrence, biosyn- thetic mechanisms, biodegradations, and industrial and medical applications of thesepolymers. Fukui,Japan YoshimitsuHamano v Contents OccurrenceandProductionofPoly-Epsilon-L-Lysine inMicroorganisms ............................................................. 1 MunenoriTakeharaandHideoHirohara BiochemistryandEnzymologyofPoly-Epsilon-L-Lysine Biosynthesis .................................................................... 23 YoshimitsuHamano BiochemistryandEnzymologyofPoly-Epsilon-L-Lysine Degradation .................................................................... 45 ToyokazuYoshida BiotechnologicalProductionofPoly-Epsilon-L-LysineforFood andMedicalApplications ..................................................... 61 KazuyaYamanakaandYoshimitsuHamano OccurrenceandBiosyntheticMechanism ofPoly-Gamma-GlutamicAcid .............................................. 77 MakotoAshiuchi EnzymaticDegradationofPoly-Gamma-GlutamicAcid .................. 95 KeitarouKimuraandZuiFujimoto PharmaceuticalandMedicalApplications ofPoly-Gamma-GlutamicAcid ............................................. 119 TakamiAkagi,MichiyaMatsusaki,andMitsuruAkashi FoodApplicationsofPoly-Gamma-GlutamicAcid ........................ 155 HiroyukiTanimoto Index ........................................................................... 169 vii Occurrence and Production of Poly-Epsilon- -Lysine in Microorganisms L MunenoriTakeharaandHideoHirohara Contents 1 Introduction ..................................................................................2 2 ScreeningandDiscoveryofPoly-e-L-LysinePolymers......................................3 2.1 FirstDiscoveryasDragendorff-PositiveSubstance....................................4 2.2 EveryProducerStrainHase-PL-DegradingActivity ..................................4 2.3 HighThroughputScreeninginAgarPlates ............................................5 2.4 FrequentOccurrencesFoundbyTwo-StageCultureMethod..........................5 3 ProductionBehaviorinStreptomycesStrains ................................................6 3.1 FeaturesShownbytheTwoStrains ...................................................7 3.2 EffectsoftheCultureMedium.........................................................8 3.3 ReleaseofPolymersintotheCultureBroth ...........................................9 3.4 Developmentofe-PL-HydrolyzingActivity ......................................... 10 4 PolymerStructureofe-PLinStreptomycesStrains ........................................ 12 5 TwoAdvantageousProducers ............................................................. 14 5.1 StreptomyceslydicusUSE-11 ........................................................ 14 5.2 StreptomycesaureofaciensUSE-82.................................................. 14 6 ProductionControlandChainLengthShortening ......................................... 15 6.1 CellDensity-DependentProduction ................................................. 15 6.2 ChainLengthShorteningbyAliphaticHydroxy-compounds ........................ 16 6.3 ChainLengthShorteningAssistedbySulfatedb-Cyclodextrin ..................... 17 7 Poly(AminoAcid)Coproducedwithe-PL ................................................. 18 7.1 Poly-g-L-DiaminobutanoicAcid ..................................................... 18 7.2 Lariat-ShapedPoly-g-L-GlutamicAcid .............................................. 19 8 ConcludingRemarks....................................................................... 20 References ......................................................................................21 M.Takehara(*) Department of Materials Science, University of Shiga Prefecture, 2500 Hassaka, Hikone 522-8533,Japan e-mail:[email protected] H.Hirohara(*) Department of Materials Science, University of Shiga Prefecture, 2500 Hassaka, Hikone 522- 8533,Japan MEALaboratory,4-31-18Takakuradai,Minami-ku,Sakai590-0117,Japan e-mail:[email protected] Y.Hamano(ed.),Amino-AcidHomopolymersOccurringinNature, 1 MicrobiologyMonographs15,DOI10.1007/978-3-642-12453-2_1, #Springer-VerlagBerlinHeidelberg2010 2 M.TakeharaandH.Hirohara Abstract This chapter addresses the occurrence and production of poly-e-L-lysine (e-PL) in filamentous bacteria from the family Streptomycetaceae and ergot fungi, especiallyinthegenusStreptomyces.Thepresenceofe-PL,firstdiscoveredfroma strainamong 2,000 actinomycetes, was found quite frequentlyin various strains of Streptomyces by novel screening methods, including the two-stage culture of cell growth and e-PL production cultures. Using the newly isolated producer strains of Streptomyces,theirproductionbehaviorswerestudiednotonlyintermsofthetime courseof several production factors and effect ofculture medium components, but alsootheraspectsofthereleaseofsynthesizede-PLintotheculturebrothandofthe simultaneous development of e-PL hydrolase activity with the e-PL-producing machinery. The e-PLs obtained were evaluated structurally. The results revealed thatthepolymershadanearlymonodispersedstructure,andcouldbeclassifiedinto five groups based on their chain lengths. The cell density-dependent control of the productionofe-PL,thechainlengthshorteningbyaliphatichydroxy-compounds,and thecoproductionofnovelaminoacidhomopolymerswithe-PLarealsodiscussed. 1 Introduction Poly-e-L-lysine(e-PL)(alsocallede-poly-L-lysine)isanL-lysinelinearhomopoly- mer biosynthesized extracellularly, and has a unique structure linking e-amino and a-carboxylic acid functional groups (Fig. 1). The polymer of 25–35 residues wasdiscoveredasasecretedproductfromastrainofStreptomycesalbulusNo.346, now designated S. albulus NBRC 14147 (NBRC 14147), in culture filtrates (Shima and Sakai 1977). The compound is biodegradable and water soluble, and has various functions such as antimicrobial activity (Shima et al. 1984; Hiraki 2000), antiphage action (Shima et al. 1982), endotoxin-selective removal action (Hirayama et al. 1999), and antiobesity action due to the inhibition of pancreatic lipase(Tsujitaetal.2006).Thispolymerispracticallynontoxicinacute,subchro- nic and chronic feeding studies in rats, and nonmutagenic in bacterial reversion assays(Hirakietal.2003).SincethediscoveryofNBRC14147,theproductionof e-PL has been enhancednearly 100-foldthroughvariousoptimization attemptsin fermentationtechniquessuchasstrictcontrolsofthepHandglucoseconcentration ofculturemediausingacertainmutantofthefirststrain(Kaharetal.2001).e-PL is manufactured at the commercial scale by a fermentation process using the mutant of NBRC 14147, and is used as a food preservative in several countries (Oppermann-SanioandSteinb€uchel2002;YoshidaandNagasawa2003). Despitethefactthatthispolymerwasscientificallysointerestingandpractically souseful,studiesone-PLhavebeenratherlimitedbothinquantity,scopeandthe O H d b Fig.1 Chemicalstructureof N a e-poly-L-lysine(e-PL) e g biosynthesizedin NH + microorganisms 3 n OccurrenceandProductionofPoly-Epsilon-L-LysineinMicroorganisms 3 level of detail examined as compared with poly-g-glutamic acid (g-PGA) (see chapter “Occurrence and Biosynthetic Mechanism of Poly-Gamma-Glutamic Acid” by Ashiuchi) or cyanophycin, the storage amino acid polymer which accu- mulates inside producing cells (Oppermann-Sanio and Steinb€uchel 2002, 2003). Thismightbemainlyattributedtothefactthateversincethefirstdiscoveryofthe S. albulus strain, no microorganisms producing e-PL had been isolated until recently when two novel screening methods succeeded inisolating several strains of Streptomycetaceae and ergot fungi (Nishikawa and Ogawa 2002; Kito et al. 2002a). All of the specific properties mentioned above were studied using e-PL samples fromNBRC 14147 or its mutant. g-PGAwas discovered40 years before e-PL(Iva´novicsandErdo¨s1937),andmanyexperimentshavebeenperformedonit over the years in various fields and levels. Cyanophycin, discovered in the nine- teenth century, has also been well studied in terms of its biosynthesis at the molecularandbiologicallevels(Oppermann-SanioandSteinb€uchel2002,2003). Under these circumstances, the presence of e-PL was found to be much more frequentthanhadbeenanticipated,throughthescreeningofvariousactinomycete strains (Hirohara et al. 2007). Of the plus 200 strains found to produce cationic polymers,tenstrainsandtheire-PLswerestudiedindetail.Alltenbelongedtothe genus Streptomyces. The authors examined the effects of the components of the culture medium on e-PL production as well as the production behaviors in these strains (Hirohara et al. 2006). They reported the number of lysine residues (R ), n number and weight average molecular weight (M , M ), and polydispersity index n w (M /M )ofthepolymersobtainedfromglycerolorglucose(Hiroharaetal.2007). w n They also studied how the e-PL was released into the culture broth, and how the development of e-PL-production and hydrolyzing activities were correlated in certain producer strains (Saimura et al. 2008). All of these reports will further facilitatethestudyofe-PLinbothfundamentalresearchandtechnicalapplications byobtainingavarietyofnovelpolymerswithdesirablepolymericstructures. Thischaptergivesanup-to-dateoverviewontheoccurrenceandproductionof e-PLinmicroorganisms.Itincludesthefrequentoccurrenceofe-PLswithvarious R s, the nearly monodispersed structures of e-PLs irrespective of their R s, the n n controlofproduction,shorteningofthechainlengththroughesterification,andthe coproduction of another amino-acid homopolymer (poly(amino acid)) with e-PL. The biosynthetic mechanism is not discussed here, since the genes and enzymes involved in the biosynthesis are discussed fully in chapter “Biochemistry and EnzymologyofPoly-Epsilon-L-LysineBiosynthesis”byHamano. 2 Screening and Discovery of Poly-«-L-Lysine Polymers A quarter of a century after the discovery of the first producer strain, a dozen microorganisms have been found to produce the polymer using two novel and independent screening methods. Thereafter, the much more frequent presence of e-PLthanhadbeenpreviouslyanticipatedwassupportedbyscreeningsofvarious
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