Enzymes in the Environment: Activity, Ecology and Applications - Chapter 15 pps

... Inc.assuchanalysesassumesampleindependence.Thisinformationisvaluableinthedesignofsamplingstrategiesforbioindicatorsasanunderstandingoftherepresentativenessofsamplesofalargerareaiscritical.Thethirdimportantfeatureisthenugget.Theoretically,whenthelagdistanceiszero(samplestakenatthesamepoint)thereshouldbenovariance.OftenthesemivariograminterceptsalongtheYaxis,notattheorigin,andthisisknownasthenuggeteffect.Presenceofanuggetindicateseithermeasurementerrororspatialstructureoverdistancesshorterthantheintervalsbetweensamplinglocations.Structuralvarianceisthefourthpropertycharacterizedbythesemivariogram.Thisvalue,whichisoftenexpressedasaratiobetweenthevariancenotexplainedbythenuggetandthetotalvariance,quantifiestheamountofvariancearisingfromtheunderlyingspatialstructure.Thegreatertheratio,themorespatiallydependentthesoilparameteris.Informationgener-atedinthevariogramisthenusedforkriging.Krigingallowsmaps(Fig.3)thatpredictparameter ... Marcel Dekker, Inc.acid proline (34) and the latter including polyamines such as spermidine and putrescine(35). The activities of certain plant enzymes, such as peroxidases and catalases, can ... that is outside the cell, and the significanceof these and other enzymes in soil microbial ecology has been reviewed (14). These extra-cellular enzymes are often relatively stable and can persist...

... 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 ... 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 ... phenylalanine, proline, methionine, and cysteine by birnessite, and the role of pyrogallol in influencing their mineralizationhave been investigated (152 ,153 ). Nitrogen mineralization was inhibited by pyrogallol,whereas...

... 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...

... 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...

... 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, ... 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 ... the cyto-plasmic membrane, where they hydrolyze macromolecules in close vicinity to the cell. The resulting low-molecular-weight products are then transported across the cell mem-brane and utilized...

... 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 ... drought on non-mycorrhizal and mycorrhizal maize: Changes in the pools of non-structural carbohydrates, in the activities of invertase and trehalase, and in the pools of amino acids and imino acids.New ... Inc.4)-glucans(53).Xyloglucansare -1 ,4-glucanswithsidechainsthatcanhydrogenbondtocellulosemicrofibrils,cross-linkingthemandrestrainingcellexpansion.Inadditiontoastructuralrole,xyloglucanscanbehydrolyzedbyhydrolyticenzymes,andtheoligosac-charidesproducedmayactassignalmolecules (15, 54).Theplantcellwallcontainsglucanasesandglycosidasesthathydrolyzexyloglucanintomonosaccharides.Endo- -1 ,4-glucanaseactivityisresponsibleforthefirststepofdegradationwherebythexyloglucanisendohydrolyzedintolargefragmentsandexo-1, 4- glucanaseactivityliberateslow-molecular-weightfractionsfromtheendsoflongpolysac-charidechains(41).TheproductionofhemicellulolyticenzymeshasbeenobservednotonlyinparasitesbutalsoinmutualisticmicroorganismssuchasRhizobiumspecies(24)andarbuscularmycorrhiza(28).Endoxyloglucanaseactivityincreasesduringgrowthanddevelopmentofroots(55).Thisactivitywasconsistentlyhigheratthebeginningofcolonizationandthelogarithmicstageofdevelopmentofmycorrhizalfungus(55).Theincreaseinfungalstructuresthatpenetratethecellwallduringthelogarithmicstageofrootcolonizationmayexplaintheincreaseinthedifferentactivitiesatthistime(56).Theevolutionofendoxyloglucanaseactivitiesinplantsparalleledthechangesintheexternalmycelium.Therewere,however,bandsofxyloglucanaseactivityinnonmycorrhizalrootsthatwereabsentinmycorrhizalroots;thatmaysuggestqualitativeinhibitionbythefungusofsomeplantactivity.Inhibi-tionofplantproteinsynthesisbyAMfungihasbeenobservedinseveralplant–AMfungiassociations(57,58).III.ENZYMESINTHEPHYSIOLOGYOFTHEASSOCIATIONA.PhosphorusUptakeItnowisestablishedthatmycorrhizalcolonizationcanenhancetheuptakefromsoilofsolubleinorganicPbyplantroots(59).Althoughparticularlyimportantinlow-Psoils,anincreasedrateofPuptakecanoccuroverarangeofsoilPlevelsevenwhenmycorrhizalgrowthresponsesnolongeroccur.TheenhancedPuptakebymycorrhizalplantsismostlikelytheresultoftheexternalfungalhyphae’sactingasanextensionoftherootsystem,therebyprovidingamoreefficient(moreextensiveandbetterdistributed)absorbingsur-faceforuptakeofnutrientsfromthesoilandfortranslocationtothehostroot(60).ExternalhyphaeofAMfungimustabsorborthophosphate(Pi)byactivetransport(59,61).TheyhaveanactiveHϩ-ATPaseintheplasmamembranethatwouldbecapableofgeneratingtherequiredproton-motiveforcetodriveHϩ-phosphatecotransport,andPcertainlyisaccumulatedtohighconcentration(62).Polyphosphate(poly-P)isamajorPreserveinmanyfungianditaccumulatesinvacuolesofAMfungi(63).Transferofmycorrhizalrootsfromlow-tohigh-Pmediaresultsinarapidaccumulationofpoly-P(64).Enzymesofpoly-Psynthesishavebeenfoundinmycorrhizaltissue(63,65).Polyphosphatekinase,whichcatalyzesthetransferoftheterminalphosphatefromATPtopoly-P,wasdetectedinbothexternalhyphaeandmycorrhizalrootsbutnotinuninfectedroots,indicatingthatpoly-Pcanbesynthesizedonlybythefungalcomponentofthemycorrhiza.AlthoughitnowseemslikelythatPistranslocatedbyprotoplasmicstreamingintotheintraradicalhyphaeaspoly-P(66),littleisyetknownofthebiochemicalmechanismsinvolved.Thetransportthroughthehyphaeandunloadingstepswithinthearbusculemaybelinkedtopoly-Pmetabolism(Fig.2).Highproportionoflong-chainpoly-PtototalCopyright...

... soil enzymes, laccase and tyrosinase. The potential role of these enzymes in the humification of anilinic and phenolic compounds and reduction of their bioavilability with the passage of time (aging) ... Inc.(nitrificationanddenitrificationeffects)aswellasbyprotectionandcreationofwetlands(4,7,39,57,85,122).Itisagainstthisbackdropofthemajorenvironmentalrelevanceoftheenzymesofnitrogenandcarboncyclingprocessesthatthischapterispresented.Theutilityofsoilenzymeactivitiesasindicatorsofsoilqualityandinmonitoringoftheeffectsofsoilpollutionispresentedelsewhere(14,34,60,116,131)andinChapters15,16 ,and1 7 .The general objective of this chapter is to highlight the current status of our understanding ofsoil carbon and nitrogen processes and the properties of the soil ... 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...

... each site and placed in litter 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, ... 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 ... everycombination of monomer, linkage, and secondary structure (69). For lignin and other aro-matic molecules, the process is principally oxidative; the enzymes have lower specificity,but the full...