Enzymes in the Environment: Activity, Ecology and Applications - Chapter 12 ppsx

... systems, lasR-lasI and rhlR-rhlI. The lasI and rhlI gene productsare involved in the synthesis of two different AHL molecules, N-(3-oxododecanoyl)-l-homoserine lactone and N-buytryl-l-homoserine lactone, ... components in- clude β-galactosidase, β-N-acetylglucosaminidase, β-N-acetylgalactosaminidase, - and β-mannosidase, and α-fucosidase (116). Other bacteria then produce proteolytic enzymes, such ... Inc.Microbialmatsareexamplesofthicklylayeredbiofilmsofphotosyntheticmicro-organismsattachedtorocksandsedimentparticlesinaqueoushabitats(25).Theyareoftenfoundunderextremeenvironmentalconditions.Forexample,inthevicinityofdeepseahydrothermalvents,microorganismswithinbiofilmssurviveextremetemperatures(86,87).HotspringsareanotherextremehabitatwherebothhightemperaturesandsulfideconcentrationsharbormatscontaininglayersprimarilycomposedofArchaea,includingsulfate-reducingpurplebacteria(e.g.,Chloroflexisspp.,Chromatiumspp.,Thiopediaro-seopersicinia)inassociationwithcyanobacteria(25).Additionalextremeenvironmentswheremicrobialmatsmaybefoundincludehypersalinelakes(88),terrestrialdesertswithcyclicaldroughtanddesiccation,sodalakesandacidthermalwaterscontainingextremepHconditions,andregionswithhighlevelsofultraviolet(UV)irradiation(88).Themicro-bialspeciesthatarefoundintheseextremeenvironmentsarelimitedtoprimarilycyano-bacteria(e.g.,OscillatoriaandSpirulinaspp.)andotherssuchasDesulfovibriospp.,Beg-giatoaspp.,andThiovulumspp.,withdifferingandvaryingdegreesoftolerance(89).Althoughmatsareprimarilycomposedofprokaryotes,otherorganisms,suchastheeukar-yoticCyanidiumsp.,havebeenfoundatpHlevelsbelow4.5(89).Studieshaveshownthatmostoftheorganismswithinamatareoftennotphysiologicallyadaptedtotheextremeenvironmentbutgrowthwithinlayersofathickbiofilmhelpsthemsurviveandfindasuitablemicroniche(89).Microbialmatsareagoodexampleoftheprotectivenatureofbiofilmgrowthandthemethodwithwhichstratificationcanencouragenutrientavailabilityandcycling(90).Biofilmshavebeenobservedatotheraquaticinterfacesbesidesthoseatasolid–liquidinterface.Forexample,instagnantwaters,biofilmsaresometimesfoundattheair–liquidinterfaceandareoftenseenasbrownorgreenlayerscomposedofalgaeandotheraquaticmicroorganisms.Anotherexampleisthewaxytypebiofilmattheair–liquidinter-faceformedfromtherugosephenotypeofVibriocholeraeisolatedfromstarvationme-dium(91).Theinterfacebetweenjetfuelsandwatercanalsoharborbiofilmgrowth,suchasthefungusCladosporiumresinae(92).VII.NONAQUATICENVIRONMENTSAlthoughbiofilmshaveoftenbeenstudiedinaquaticenvironments,morerecentstudieshaveshownthatmicroorganismswithinthickEPSmatricesorbiofilmsarealsofoundinnonaquaticenvironmentssuchastherhizosphere (Chapter4 ),soil,andsubsurfaceenviron-ments(93,94).Oneofthemorecomplexenvironmentsisthesoilecosystem,withitsmanydifferentparticlesandporespaces(95).Microorganismsinthesoiladheretosurfacessuchasinorganicsolidparticles,humicmatter,plantmaterial(roots),andmicrofauna.Plantsprovidelargeamountsofcarbonandothernutrientstoencouragemicrobialgrowthinthevicinityoftheroots ,and, inturn,themicroorganismsfixnitrogen,assisttheplantinadsorptionofnutrientsfromthesoil,andprotecttherootsagainstpathogens.Anotherexampleofanonaquaticbiofilmisthecolonizationoftheleavesofplants—thephyllo-sphere(96 ;Chapter6 ).Thesebiofilmsconsistofadiversepopulationofmicroorganisms,including...

... systems involving phenoloxidase enzymes. The deamination of amino acids, such as serine, phenylalanine, proline, methionine, and cysteine by birnessite, and the role of pyrogallol in influencing their ... effective for both L- and D- glutamic acid. The PLP-Cu2ϩ-smectitehas acted as a ‘‘pseudoenzyme’’ wherein the PLP was active and independent of the protein matrix of the enzyme and the silicate structure ... aspartase-Ca-montmoril-lonite systems (159). Deamination of l- and d-glutamic and aspartic amino acids and oftheir DL racemic mixtures in the presence of Na-montmorillonite showed a stereoselectiv-ity...

... Inc.possibletofindseveralexplanationstointerpretaproteinadsorptionisotherm,withnoexperimentalevidenceavailabletochooseamongthem.TheadvantageoftheNMRmethodisthatitsimultaneouslygivesthequantityofadsorbedprotein,thesurfacecover-ageofthesolidbytheprotein,andthemonolayerormultilayermodeofadsorption(16).Onlyknowledgeofthesethreefactorsallowsapossibleunfoldingoftheproteinsontheclaysurfacestobedetectedandquantified.1.NuclearMagneticResonanceDetectionoftheExchangeofaParamagneticCationonProteinAdsorptiononClaysTheprincipleofthemethod(16)isbasedonthefactthattheadsorptionofproteinsonclayscausesthereleaseofcharge-compensatingcations(7,17).ItalsousesthesensitivityoftherelaxationtimesT1andT2ofnuclearspinstoparamagneticcationsinNMRspectros-copy(18,19).Asmallquantity(between 3and2 0µMdependingonthepH)ofaparamagneticcation,Mn2ϩ,isaddedtoasodium-saturatedmontmorillonitesuspension(1gLϪ1)witha10-mMconcentrationoforthophosphate.Thesuspensionisstudiedby31PNMRspec-troscopy.Aninterestingphenomenonisobserved:(1)theMn2ϩcationsthatareadsorbedontheclaysurfacedonotinteractatallwiththeorthophosphate,asshownbythecompari-sonbetweentheclaysuspensionandsupernatantafterremovaloftheclaybycentrifuga-tion ;and( 2)theMn2ϩcationsinsolutioninteractwiththeorthophosphate,leadingtoalinearincreaseofthelinewidthathalfheight,∆ν1/2,oftheorthophosphatepeakontheNMRspectrum.Thislasteffectistheresultoftheparamagneticcontributiontothede-creaseofthespin–spinrelaxationtime,T2,oftheorthophosphatesignal.Whenagivenquantityofproteinisintroducedintothissuspension,itdisturbstheequilibriumbetweentheparamagneticMn2ϩadsorbedontheclaysurfaceandthatinsolution.Theanalysisoftheresultinglinewidthoftheorthophosphosphatesignalgivesthequantityofcationsexchangedonadsorption.Witha300-MHzNMRspectrometer,themeasurementtakesafewminutes;witha500-MHzspectrometer,1minissufficient(evenlessifhigherconcentrationsofortho-phosphateareused).Asnocentrifugationisrequiredwiththismethod,thisshorttimeofsignalacquisitioniscompatiblewithkineticstudies.Theresultsareexpressedas∆νP,whichisthedifferencebetween∆ν1/2inthesystemwithparamagneticcationsand∆ν1/2inacontrolofthesamecomposition,(butwithoutparamagneticcations)dividedbytheconcentrationofparamagneticcations.ThesurfacecoverageoftheclaybytheproteincanbededucedfromthefractionofMn2ϩreleased.Theknowledgeofboththequantityofproteinadsorbedandthesurfacecoverageofthesolidallowsthecalculationoftheinterfacialareaofcontactbetweenasingleproteinmoleculeandtheclaysurfaceatdiffer-entpHandionicstrengths.2.ConformationalChangesonAdsorptionofaSoftProtein,BovineSerumAlbumina.DescriptionoftheProgressiveSurfaceCoverageoftheClayFigure1shows the ... (2) a possi-ble unfolding of the protein on the surface changing the interfacial area between individualprotein and surface and the quantity of protein adsorbed at saturation; (3) the surfacecoverage ... contrast to the three preceding models, which assume that the enzymes retain the sameconformation in the adsorbed state and in solution, another model is based on a pH-depen-dent unfolding of the enzyme...

... Inc.Currently,itisevidentthatmicroorganismsformcomplexmicrobialfoodwebsinallaquaticecosystems,andthattheiractivitiesandmetabolismsoftenaretightlycoupled and/ ormutuallyaffected(132,143,144).Therefore,itisnotsurprisingthatenzymaticpropertiesandactivitiesofdifferentcomponentscreatingthemicrobialfoodwebsinlakeecosystemshavedemonstratedcloserelationships.Severalreportshavedocumentedthestrongdependencyofbacterialsecondaryproductiononectoenzymeactivitiesofaquaticmicroorganisms(2–4,16,17,19,25,28,29,33,36,59).Thereoftenisasignificantcorrelationbetweenphytoplanktonprimaryproductionandactivitiesofdifferentectoenzymesinfreshwaterecosystems(25,28,29,33,52).Ourstudiesinlakesofdifferingdegreesofeutrophicationhaveshownmicrobialesteraseactivitytobepositivelycorrelatedtophytoplanktonprimaryproduction,bacterialsecondaryproduction,andconcentrationofdissolvedorganiccarbon(DOC)(Fig.13).Wehavefoundasignificantnegativerelationshipbetweenenzymeactivityandtheper-centageofphytoplanktonextracellularrelease(PER)ofphotosyntheticorganiccarboninthestudiedlakes.ThisnegativecorrelationbetweenPERandesteraseactivityindicatedthatenzymesynthesiswaspartiallyinhibitedinbacteriabylow-molecular-weightphoto-syntheticproductsofphytoplanktonthatwerereadilyutilizedbythesemicroheterotrophs:i.e.,catabolicrepressionofesterasesynthesiswasfoundinlakescharacterizedbyhighPERofphytoplankton(29,33).VIII.ECTOENZYMEACTIVITYANDLAKEWATEREUTROPHICATIONTheimportanceoforganicmatterasavariableforevaluatingthetrophicstatusoflakeshasbeenrecognizedsincethebeginningofthe20thcentury(145,146).Increasingconcen-trationsoforganicconstituentsinwaterarethedistinctindicatorsofacceleratedeutrophi-cationprocessesinmanylakes(147–149).OurstudiesclearlydemonstratedthatenzymeactivitiesweresignificantlypositivelyproportionaltoDOCcontentoflakes(Fig.13C).Asdescribedearlierinthischapter,severalmicrobialectoenzymesareresponsibleforrapidtransformationanddegradationofbothdissolvedorganicmatterandPOMinfresh-waterecosystems.Therefore,wehypothesizethatan‘‘enzymaticapproach’’canbeveryusefulinthestudiesoflakeeutrophication.Severalreportspointedoutthatmicrobialenzymaticactivitieswerecloselyrelatedtotheindicesofwatereutrophicationand/orthetrophicstatusofaquaticecosystems(25,27,29,31,33,38,52,58,62,78).Ourstudiesalongthetrophicgradientoflakes(fromoligo/mesotrophictohypereutrophiclakes[Fig.14A]supportourhypothesis(andtheassumptionsofothers)thatselectedenzymaticmicrobialactivitiesareverypracticalforarapidrecognitionofthecurrenttrophicstatusoflakes.Activitiesofalkalinephosphatase,esterase,andaminopeptidaseincreasedexponentiallyalongatrophicgradientandcorre-latedsignificantlywiththetrophicstateindexofthestudiedlakes(Fig.14B,C,D).Wealsofoundastrongrelationshipbetweenactivitiesofectoenzymesandphytoplanktonprimaryproductionintheselakes.RapidincreasesinectoenzymeactivitieswereobservedespeciallyinarangeofgraduallyeutrophiclakeswhenthevalueofCarlson’strophicstateindex(TSI)wasabove55(150)(Fig.14).Moreover, ... for the enzymes involved in the transformation and degrada-tion of polymeric substrates outside the cell membrane: ectoenzymes (19), extracellular enzymes (20), and exoenzymes (21). In this chapter, ... Inc.lakewater.Figures2Band2CshowthatectoenzymesynthesisinDOM-enrichedsampleswasnolongerrepressedwhentheconcentrationofthereadilyutilizablelowmolecular-weightmoleculesfellbelowacriticallevel,andpolymericsubstrateshadtobeusedtosupportthegrowthandmetabolismofbacteria.Similarinsituobservationsduringphyto-planktonbloomdevelopmentandbreakdownwerereportedforβ-glucosidaseactivityineutrophicLakePlußsee(24),forβ-glucosidaseandaminopeptidaseactivitiesinmeso-trophicLakeScho¨hsee(25),andforlipaseactivityineutrophicLakeMikołajskie(40).Despitethewidespreadoccurrenceofcatabolicrepression,withtheexceptionofthoseforentericbacteria,themoleculardetailsoftherepressionarepoorlyunderstood.Somestudieshaveindicatedthatcyclicadenosinemonophosphate(cAMP),togetherwithitsreceptorprotein,mayplayacentralroleincontrolofcatabolicrepression(41,42).Usingtherepressionstrategyforectoenzymesynthesis,microorganismscanavoidthewastefulproductionofinducibleenzymes,whicharenotusefulwhentheirgrowthisnotlimitedbyUDOM(3,19,24,35).B.InhibitionofActivityItisimportanttoconsiderthattherepression/derepressionofanectoenzymenotbeequatedtothereversibleinhibitionofactivity.Evenifanectoenzymeissynthesized,itsactivitymaybeinhibitedbytheaccumulationoftheendproductorbyhighconcentrationsofthesubstrate(19).Twogeneraltypesofreversibleinhibitionareknown:competitiveandnoncompetitiveinhibition.Competitiveinhibitionoccurswhenaninhibitingcompoundisstructurallysimilartothenaturalsubstrateand,bymimicry,bindstotheenzyme.Indoingso,itcompeteswithanenzyme’snaturalsubstratefortheactivesubstrate-bindingsite.Thehallmarkofcompetitiveinhibitionofmanyectoenzymes(e.g.,alkalinephosphatase,β-glucosidase,aminopeptidase)isthatitdecreasestheaffinityofanectoenzyme(anincreaseoftheapparentMichaelisconstantisobserved)forthesubstrateand,therefore,inhibitstheinitialvelocityofthereaction(Fig.3)(13,26,37).Competitiveinhibitionisreversibleandcanbeovercomebyincreasedsubstrateconcentration,andthereforethemaximumvelocity(Vmax)ofthereactionisunchanged(Fig.3A).Noncompetitive...

... forchitin-hydrolyzing activity by using MUF-β-d-N, N′-diacetylchitobioside, and chitobiaseactivity was then assayed in protein extracts prepared from the positive clones. The chi-tinases of marine bacteria ... Inc.Investigationsofextracellularenzymesfrommarineanimalsandenzymesisolatedfromprokaryotesareconsideredonlyifaclearconnectiontomarineecologyisestablished.Thetermextracellularenzymesisusedthroughoutthischapter,whereasChro´st(5)distin-guishesbetweenectoenzymesandextracellularenzymes.EctoenzymesaredefinedbyChro´st(5)andinChapter2asenzymeslocatedintheperiplasmicspaceorattachedtotheoutermembraneofthebacterialcell.Extracellularenzymesareenzymesfreelydis-solvedinthewaterorattachedtoparticlesotherthantheenzyme-synthesizingcell .In thischapter,however,thetermextracellularenzymesreferstobothectoenzymesandextracellularenzymes,unlessotherwisestated.EarlystudiesonthefateoforganicaggregatesanddissolvedpolymersintheseawerepresentedbyRiley(6),Walsh(7),andKhailovandFinenko(8).Overbeck(9)re-viewedtheearlystudiesonextracellularenzymeactivityintheaquaticenvironment.II.ECOLOGICALPRINCIPLESOFENZYMATICPATTERNSINTHESEAA.TheConceptoftheMicrobialLoopandtheRoleofExtracellular Enzymes Themicrobialloop(10)encompassesthecombinedactivitiesofautotrophicandheterotro-phic—eukaryoticaswellasprokaryotic—organismssmallerthan20µm.Theseorgan-isms,representedbybacteria,nanoflagellates,ciliates,andphototrophicprochlorophytes,aswellascyanobacteria,formafoodweboftheirown,looselyconnectedtothefoodwebofthelargergrazers.Ingeneral,thenutritionalbasisofthemicrobialfoodwebisprovidedbythepoolofdissolvedorganicmatter(DOM)andparticulateorganicmatter(POM).TheDOMpoolisapriorireservedforbacterialutilization,whereascompetitionwithmetazoansoccursforPOM.ThiscompetitionisdeterminedbythebacterialpotentialforenzymaticdissolutionofPOMontheonehandandthefeedingactivityofthemetazo-ansontheotherhand.Thebulkofboththedissolvedandparticulateresources,however,requiresenzymatichydrolysispriortouptakebybacteria(Fig.1).Thustheenzymaticactivitiesofbacteriainitiateorganiccarbon(C)remineralizationanddefinethetypeandquantityofsubstrateavailabletothetotalmicrobialfoodweband,tocertainextent,alsotothetoppredatorsinthesystem.B.FreeandAttachedEnzymeActivityGenerally,extracellularenzymesmaybeboundtothecell(definedasectoenzymesbyChro´st[5])orinthefreeandadsorbedstate(11 ,12) .Mostofthetotalenzymeactivityinseawaterhasbeenfoundtobeassociatedwiththeparticlesizeclassdominatedbybacteria(Ͼ0.2µm–3µm)(13,14)(Table1).Dissolvedenzymes(15)andlargeparticlesϾ8 ... Inc.Investigationsofextracellularenzymesfrommarineanimalsandenzymesisolatedfromprokaryotesareconsideredonlyifaclearconnectiontomarineecologyisestablished.Thetermextracellularenzymesisusedthroughoutthischapter,whereasChro´st(5)distin-guishesbetweenectoenzymesandextracellularenzymes.EctoenzymesaredefinedbyChro´st(5)andinChapter2asenzymeslocatedintheperiplasmicspaceorattachedtotheoutermembraneofthebacterialcell.Extracellularenzymesareenzymesfreelydis-solvedinthewaterorattachedtoparticlesotherthantheenzyme-synthesizingcell .In thischapter,however,thetermextracellularenzymesreferstobothectoenzymesandextracellularenzymes,unlessotherwisestated.EarlystudiesonthefateoforganicaggregatesanddissolvedpolymersintheseawerepresentedbyRiley(6),Walsh(7),andKhailovandFinenko(8).Overbeck(9)re-viewedtheearlystudiesonextracellularenzymeactivityintheaquaticenvironment.II.ECOLOGICALPRINCIPLESOFENZYMATICPATTERNSINTHESEAA.TheConceptoftheMicrobialLoopandtheRoleofExtracellular Enzymes Themicrobialloop(10)encompassesthecombinedactivitiesofautotrophicandheterotro-phic—eukaryoticaswellasprokaryotic—organismssmallerthan20µm.Theseorgan-isms,representedbybacteria,nanoflagellates,ciliates,andphototrophicprochlorophytes,aswellascyanobacteria,formafoodweboftheirown,looselyconnectedtothefoodwebofthelargergrazers.Ingeneral,thenutritionalbasisofthemicrobialfoodwebisprovidedbythepoolofdissolvedorganicmatter(DOM)andparticulateorganicmatter(POM).TheDOMpoolisapriorireservedforbacterialutilization,whereascompetitionwithmetazoansoccursforPOM.ThiscompetitionisdeterminedbythebacterialpotentialforenzymaticdissolutionofPOMontheonehandandthefeedingactivityofthemetazo-ansontheotherhand.Thebulkofboththedissolvedandparticulateresources,however,requiresenzymatichydrolysispriortouptakebybacteria(Fig.1).Thustheenzymaticactivitiesofbacteriainitiateorganiccarbon(C)remineralizationanddefinethetypeandquantityofsubstrateavailabletothetotalmicrobialfoodweband,tocertainextent,alsotothetoppredatorsinthesystem.B.FreeandAttachedEnzymeActivityGenerally,extracellularenzymesmaybeboundtothecell(definedasectoenzymesbyChro´st[5])orinthefreeandadsorbedstate(11 ,12) .Mostofthetotalenzymeactivityinseawaterhasbeenfoundtobeassociatedwiththeparticlesizeclassdominatedbybacteria(Ͼ0.2µm–3µm)(13,14)(Table1).Dissolvedenzymes(15)andlargeparticlesϾ8...

... Inc.Althoughthisstudyinvolvedtheuseofageneticallymodifiedmicrobe,themodi - cationswerenotintendedtohaveafunctionalimpact;theywereinsertedasgeneticmark-ers.Asecondstudycomparingtheeffectofthesamegeneticallymarkedstraintothatofafunctionallymodifiedstrainshowedeffectsthataremoreinteresting(36).Theaimofthisworkwastodeterminetheimpactintherhizosphereofwildtypealongwithfunction-allyandnonfunctionallymodifiedPseudomonasfluorescensstrains.Thewild-typeF113straincarriedageneencodingtheproductionoftheantibiotic2,4-diacetylphloroglucinol(DAPG),usefulinplantdiseasecontrol,andwasmarkedwithalacZYgenecassette .The firstmodifiedstrainwasafunctionalmodificationofstrainF113withrepressedproductionofDAPG,creatingtheDAPGnegativestrainF113G22.Thesecondpairedcomparisonwasanonfunctionalmodificationofwild-type(unmarked)strainSBW25,constructedtocarrymarkergenesonly,creatingstrainSBW25EeZY-6KX.Significantperturbationswererecordedintheindigenousbacterialpopulationstruc-ture;theF113(DAPGϩ)straincausedashifttowardslower-growingcolonies(Kstrate-gists)comparedwiththenon-antibiotic-producingderivative(F113G22)andSBW25strains.TheDAPGϩstrainalsosignificantlyreduced,incomparisonwiththoseoftheotherinocula,thetotalPseudomonassp.populations,butdidnotaffectthetotalmicrobialpopulations.ThesurvivalofF113andF113G22wasanorderofmagnitudelowerthanthatoftheSBW25strains.TheDAPGϩstraincausedasignificantdecreaseintheshoot-to-rootratioincomparisontothatofthecontrolandotherinoculants,indicatingplantstress.F113increasedsoilalkalinephosphatase,phosphodiesterase,andarylsulfataseac-tivities(Table2)comparedtothoseofthecontrols.Theotherinoculareducedthesameenzyme ... Inc.Theresultsshowedlargedifferencesbetweenthe2daysofsamplinginsoilenzymeactivities(e.g.,alkalinephosphatase,Fig.2)andavailablesoilnutrients(e.g.,nitrate,Fig.3).Differenceswerefoundalsobetweenthevariousoilseedrapevarietieswithmostsoilenzymesmeasuredandwiththeavailablesoilnutrients.However,therewaslittlediffer-encebetweentheenzymeactivitiesintherhizosphereoftheGMandnon-GMplants.Themajorfactorinfluencingtheenzymeactivitiesandsoilnutrientsbetweenthetwosamplingdayswasthesoilmoisturecontent,whichwasincreasedbyovernightrain.Therefore,inthisfieldtrial,thedifferencesbetweensoilenzymeactivitieswerenotattrib-utabletoplantgeneticmodification,buttoenvironmentalvariationandtodifferencesinplantvariety.V.CONCLUSIONSClearlyenzymeactivitiesareusefulindeterminingperturbationsinthesoilenvironmentbroughtaboutbychangesinagriculturalpractices,theuseofagrochemicals,pollutionevents,ortheexploitationofgeneticallymodifiedorganisms.Biocontrolofpestsanddiseasesisameansbywhichenzymefunctionhasbeenexploited(43),butthereisevengreateropportunitytomonitorandmanipulateenzymesasgenerationsofplantnutrients,plant-growth-promotingagents,soilstructurestimulants,andbioremediationcatalysts.Althoughbioremediationhashadlessattentionthanbiocontrol,thepotentialforexploitationisenormous(44).Mostresearchhasbeenfocusedonmicrobialinoculants(bioaugmentation),butitisequallyrelevanttoconsiderhowtooptimizethefunctionoftheindigenousorganisms(biostimulation).Phytoremediation,byplantrootsthemselvesorassociatedmicrobiota(rhizoremediation),isbecominganincreasinglyinterestingcleanupsolutionforsoils.Mostattentionhasbeenpaidtoheavymetaldecontamination ,and whereasthereisinevitablysomeenzymeinvolvement,littlehasbeencharacterized.How-ever,rhizospheremicroorganismsproduceenzymesthathavethecapacitytocatabolizeawiderangeoforganicpollutants.MicrobialdehalogenationisdescribedindetailinChapters1 8and1 9,butofspecialinterestarehydrogencyanideandothernitriles.Notonly ... would in- crease the microbial P demand.Inverse trends were found with the C and N cycle enzymes in comparison to the general trend found in the P and S cycle enzymes. The F113 (DAPGϩ) strain was...

... short-chain poly-P was higher in the internal hyphae (67). Long-chain poly-P seems to be more efficient in transporting Pi from the extraradical to the intraradical part of the fungi. Activity of enzymes ... Inc.directlycontributetoreductionofpathogenviabilityandgrowth.Inaddition,theyhavebeenproposedasmediatorsinpathwaysleadingtodefense-relatedgeneexpression(136).ThereleaseofAOSinsomeplant–pathogeninteractionscanresultindamagetothehosttissues.Therefore,mechanismsthatlimitthedurationoftheoxidativeburstanditstoxiceffectsarenecessarytominimizedamagetotheplantitself.Oneofthesemecha-nismsistheactionofendogenousantioxidantenzymes,suchassuperoxidedismutases,catalases,peroxidases,andglutathioneperoxidases,whicharecapableofneutralizingtheAOS.Duringtheestablishmentofacompatibleplant–fungusAMsymbiosis,thehostplantshowedlittlereactionatthecytologicalleveltoappressoriumformationorinfectionhyphae.Occasionallysomethickeningwasobservedinepidermalcellwallsatthepointofcontactwithappressoria(105),andonlyaresponsesimilartoHRhasbeendetectedinRiT-DNA–transformedrootsofalfalfacolonizedbyGigasporamargarita(137).Nev-ertheless,recentstudies,usingthediaminobenzidine(DAB)stainingtechnique,revealedthatabrownishstain,indicativeofH2O2accumulation,waspresentwithincorticalrootcellsinthespaceoccupiedbyclumpedarbusculesandaroundhyphaltipsattemptingtopenetraterootsofMedicagotruncatulacolonizedbyG.intraradices(138).TheseresultssuggestthatalocallyrestrictedoxidativeburstcouldbeinvolvedintheresponseoftheplanttoAMformationanddevelopment.Relativelyfewdataexistconcerningthepossibleparticipationofantioxidanten-zymesintheplantresponsetoAMformation.Apeakofcellwall–boundperoxidasewasobservedduringtheinitialstagesoffungalpenetrationinleek(Alliumporrum)cells.Onceinfectionwasestablished,theactivitydecreasedtothelevelsshowninnonmycorrhizalplants(139).Inpotatoroots,theactivityofextracellularperoxidaserecoveredinrootleachateswasnotstimulatedbyAMinfection;peroxidaseactivitypergramoffreshweightwassignificantlyenhancedinAMroots(140).WhenpotatoplantsweregrownwithhigherPsupply,extracellularperoxidaseactivityincreasedlinearlywithincreasingPsupply,suggestingaroleofperoxidaseinlimitingAMinfectioninwell-P-nourishedplants(140).Theanalysisofcatalaseandascorbateperoxidaseactivitiesduringtheearlystageoftobacco–Glomusmosseaeinteractionrevealedtransientenhancementsofbothenzymaticactivitiesintheinoculatedplants(141).Theseincreasescoincidedwiththestageofappre-ssoriaformationonrootsurfacesandtheappearanceofapeakofaccumulationoffreesalicylicacidininoculatedroots(141).Thesedataindicatetheactivationofcatalaseandperoxidaseactivitiesinrootcellswherethefungusformingappressoriamightbepartoftheplantresponsetotheinvadingfungus.Theroleoftheseenzymesinthisresponsecouldberelatedtoactivationofadefensivemechanismortoaprocessofcellwallrepairatthesiteofinfection(Fig.3).Alternatively,theearlyactivationofcatalaseandperoxidasemay ... Inc.ThepatternsofenzymeactivityandmRNAaccumulationsuggestthatchitinases and -1 ,3-glucanasesmightbepartoftheearlydefenseresponsebytheplanttotheinvad-ingfungus,whichisthensuppressedassymbioticinteractionsdevelop.Inthiscontext,planthydrolasesmaybeinvolvedintheregulationofAMdevelopment.Nevertheless,someexperimentaldatarevealedthatitisnotlikelythatplantchitinasesandglucanasesareessentialtothecontrolofthegrowthofAMfungi.TransgenicplantsconstitutivelyexpressinghighlevelsofdifferentacidicformsoftobaccoPRs(includingchitinasesand -1 ,3-glucanases)becamenormallycolonizedbytheAMfungi (122 ,123 ).Thefactthatchitinasesand -1 ,3-glucanasesinducedbytheAMsymbioticfungiorbyconstitutivegeneexpression,donotpreventrootcolonizationsuggeststhattheyareineffectiveincontrollingfungaldevelopment.ThelowenzymaticaffinityforAMfungalcomponentsorinaccessibilityoftheseenzymestofungalcellwallcomponentsmaycausethisineffec-tiveness( 112) .Conversely,specificacidicformsofchitinaseand -1 ,3-glucanaseareactivatedinseveralplantscolonizedbyAMfungi.Thesesymbiotic,specificisoenzymeshavebeenreportedinpea (124 ),tobacco(118),andtomato (125 127 )rootsandaredifferentfrompathogen-inducedisoformsorconstitutiveenzymes.Inaddition,newchitosanaseisoformshavebeenshowninpea (128 )andtomato (126 ).Chitosanasesarehydrolyticenzymesactingonchitosan,aderivativepartiallyorfullydeacetylatedofchitin (129 ).Interestingly,themycorrhizal-relatedchitinaseisoformdescribedintomato-colonizedrootsappearedtodisplaychitosanaseactivity.Thisbifunctionalcharacterwasnotfoundfortheconstitutive enzymes, orinPhytophthorasp.–inducedchitinases (126 ).Mycorrhizal-specificplantchi-tinasesarenotactiveinpathogen-infectedroots(118 ,124 125 )orinRhizobiumsp.legumesymbiosis(130),indicatingadifferentialinductionandfunction.AlthoughtheprecisefunctionofplanthydrolaseactivitiesintheestablishmentofAMsymbioticinteractionisstillunclear,theirstimulationseemstobeakeypointinthemechanismofrecognitionandsignalingbetweenplantrootsandAMfungi.AregulatoryroleoftheseenzymesduringestablishmentofAMandotherrootsymbiosishasbeenproposed.Stimulationofspecificplantchitinaseshasbeenreportedinsoybean/Rhizobiumsp.(131)andectomycorrhiza(132).Ithasbeenpostulatedthatchitinasesmaybeinvolvedintherecognitionoftherhizobialnodulationsignalsand,thus,intheregulationofthenodulationprocess(133).Thedatasuggestaspecificrolefortheseenzymes,onethatcouldberelatedintheAMsymbiosistothedetection,modification ,and/ orreleaseofchitinorchitosanoligomersfromthefungalcellwallthatcanactassignalingcompoundsduringthedevelopmentofAM(Fig.3).Inthisprocessofsignalexchange,themodulationof...

... affecting the efficiency of interaction of the substrate and enzyme molecules. In other words, a portion of the enzyme molecules existing in the field soil may not be actively engaged in catalyzing their ... transforma-tions include the effect of bonding of β-d-glucosidase to a phenolic copolymer of l-tyro-sine, pyrogallol, or resorcinol (108) and of linking of urease to tannic acid (49,52). Sarkar and ... ofurease and invertase in the early 1990s. Gianfreda and coworkers (50) examined the inter-action of invertase (β-fructosidase) with montmorillonite, aluminum hydroxide, and alu-minum hydroxide–montmorillonite...

... Inc.wererepressedbyaddedN;formapleandoak,theseactivitiesincreased.Theresultssuggestedthatwhiterotfungi,whichproduceligninasesinresponsetolowNavailability,weredisplacedbysupplementalN,slowingthedecompositionofrecalcitrantlitter.HenriksenandBreland(27)alsofocusedontheroleofNinthedecompositionprocess.Usingamicrocosmsystemofwheatstrawandsoil,theyfoundthatcarbonminer-alization,fungalbiomass,andactivitiesofcellulolyticandhemicellulolyticenzymesde-creasedwithNavailability.Intheareaofcomparativeecosystemstudies,Sinsabaughetal.(62,63)followedmassloss,NandPimmobilization,andactivityof11typesofextracellularenzymesforbirchsticks(Betulapapyfera)decomposingateightupland,riparian,andloticsitesoverafirst-orderwatershed.Masslossratesamongsitesvariedbyafactorof5andwerecorrelatedwithlignocellulaseactivities.Incontrast,relationshipsbetweenmasslossandactivitiesofacidphosphataseand -1 ,4-N-acetylglucosaminidasevariedwidelyamongsites.TheserelationshipsalongwithanalysesoftheNandPcontentofthestickssuggestedthatdifferencesinmasslossratesamongsitesweretiedtodifferencesinnutrientavail-ability.Inanotherexperiment,litterbagscontainingsenescentleavesofAgeratumconi-zoidesandMallotusphilippinensiswereplacedonthefloorofayoungtropicalforestsiteinnortheastIndia(38).OtherlitterbagscontainingleavesofHolarrhenaantidysentericaandVitexglabratawereplacedatamaturetropicalforestsite.Athigher-elevationsubtrop-icalsites,litterbagscontainingPinuskesiyaandMyricaesculentaleaveswereplacedinayoungforestandbagscontainingPinuskesiyaandAlnusnepalensisleaveswereplacedinamatureforest.Sampleswereanalyzedformassloss,bacterialandfungalnumbers,cellulosecontent,Ncontent,solublesugarcontent,andactivitiesofcellulase,amylase,andinvertase.Cellulaseandamylaseactivitieswerecorrelatedwithmicrobialnumbers.Invertaseactivitycorrelatedwithsolublesugarcontent.Enzymeactivitiesandmasslossrateswerehigheratthelowerelevationsitesbutwerenotrelatedtostandage.Inasimilarstudy,thedecompositionofPinuskesiyaandAlnusnepalensisatadisturbedroadsideforestsitewascomparedwiththatatanundisturbedsite(30).Againcellulaseandamylaseactivitieswerecorrelatedwithmicrobialnumbers,whereasinvertaseactivitywaslinkedtosolublesugars.DillyandMunch(18)studiedenzymeactivitiesandmicrobialrespirationforAlnusglutinosa(blackalder)leavesdecomposingatwetanddrysiteswithinafenforest.Masslossratesweremorethantwiceasfastatthewetsite.Microbialbiomassandrespirationdecreasedovertime(16to2.3µmolgϪ1hϪ1),buttheefficiencyofCutilizationincreased.Thesetrendswereparalleledbydecreasingβ-glucosidaseactivityandincreasingproteaseactivity.III.COMPARATIVEANALYSESInthecontextofthesuccessionalloopmodel(Fig.1),therearethreedimensionsforcomparing ... bags. In using the ap-proach of Sinsabaugh et al. (73) and Jackson et al. (29), confined and in situ POM sampleswere assayed monthly for β-glucosidase, β-N-acetylglucosaminidase, β-xylosidase,phenol ... xylanase, β-glucosidase)were the least affected, phosphatase and sulfatase the most affected; N-acquiring enzymes (urease) were intermediate. Another study of heavy metal contamination in grassland...

... the years; these include vanilin, indulin, ferrulic acid, and, most importantly,14C-labeled synthetic lignins. Various fungal enzymes are involved in lignin degradation, including lignin peroxidase, ... strains and the extrac-tion of enzymes, provide complementary information on enzyme production by emphasi-zing the potential of the living hyphae and the sum of past and present activities re-spectively. ... enzymes in the upper part of the profile couldbe due to the presence of fungi (chitin in the cell walls) and arthropods (chitin in the exoskeleton) serving as substrates.Enzyme determination using...