Enzymes in the Environment: Activity, Ecology and Applications - Chapter 20 doc

... Inc.ensurestheremovalofHgfromtheenvironmentthroughatmosphericdissipation.CurrentstudiesarenowfocusingonbiologicalreductionandmethylationreactionsasaremedialapproachtoimmobilizeHg.A.ReductionofMercury(II)NumerousmicroorganismsavoidHgtoxicitybyreducingionicHg(Hg2ϩ)tovolatileHg0,apotentiallyusefulapplicationtoremoveHgfromHg-contaminatedwater.ThereductionofHg2ϩtoHg0canbemediatedbyanumberofmicroorganisms,includingentericbacteria,Pseudomonassp.,Staphylococcusaureus,Thiobacillusferrooxidans,Streptomycessp.,andCryptococcussp.(121).TheabilityofbacteriatoreduceHg2ϩislinkedtoHgresis-tance(mer)operons(122).Thehypothesizedplasmid-mediateddetoxificationmechanismisshowninFig.6.Theplasmidcodesforaprotein(merP)thatinitiallybindstoHg2ϩ in the periplasm. The Hg2ϩis then transported through the inner membrane to the cytoplasmby the membrane-bound protein merT. In the cytoplasm, ... CN-oxidizing, and Se-reducing microbes combined and immobi-lized in calcium alginate beads. Tests were conducted in single-pass 1 -in- diameter columnswith a retention time of 9 to 18 hours. The system ... Another cell-free system was used to treat mining processsolution containing cyanide and Se. The system contained cell-free extracts of P. pseudoal-caligenes, P. stutzeri, CN-oxidizing, and...

... 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...

... Inc.mouswiththermaloptimarelevanttoanorganismdependent(52)onafluxofdissolvedcompoundsfromenzymesalreadyreleasedandfunctioningoverlongerperiodsintheenvironment.Forexample,notetheshiftinthermalactivityoptimaforcold-adaptedprote-asesfrom13°Cto10°Cto8°Casafunctionofincreasingholdingtime(Fig.3).Wealsosuggestthatthefurtherexploratorystudyofmicrobialenzymesproducedinenvironmentscharacterizedbysharpthermalgradientsmayyieldenzymeswithbothhighcatalyticactiv-ityandlonglifetimesatextremetemperatures(hotorcold),acombinationoffeaturesthatsofarhasbeenobservedonlyasaresultofgeneticengineering(describedlater )and apparentlynotofevolutionarypressuresinnature.Thetemporallyandspatiallyfluctuatingthermalgradientswithinsulfidestructuresandseaicemayhaveprovidedthenecessaryselectivepressure.V.STATUSOFTHESEARCHFORHYPERTHERMOPHILICMICROORGANISMSANDENZYMESA.FocusonCulturableHyperthermophilesAlthoughthediscoveryofhyperthermophilicmicroorganismsatmarinehydrothermalventswasreportedin1982(53,54),theirpotentiallyexcitingactivitiesinsituhavebeenstudiedbyfewandremainpoorlyconstrained(1,28).Theinsituactivitiesofenzymesthathyperthermophilesmayreleaseintotheirsurroundingsarecompletelyunknown.Thisgenerallackofecologicalinformationonthefunctioningofeitherhyperthermophilicor-ganismsorenzymesintheirnaturalsettingsstandsincontrasttowhatisknownaboutorganismsandenzymesattheotherendofthetemperaturespectrum(seeSec.V.B);marinepsychrophileshavebeenknownandstudiedforalmostacentury,muchoftheworkecologicallymotivatedfromtheoutset(14).Perhapsbecauseoftheimmediaterecog-nitionofpracticalapplicationsforneworganismsfunctionalateverhighertemperatures(11,55),researcheffortsnowongoingworldwidehavefocusedheavilyonorganismandenzymeperformanceundercontrolledlaboratoryconditions,withspecificbiotechnologi-calorindustrialgoalsmotivatingthechoiceoforganism,enzyme,ortestconditions .The desiretoachieveafundamentalunderstandingofthebiochemical,metabolic,andgeneticbasisforhyperthermophilyhasoftenbeenpresentedasabettermeanstomanipulatestrainsandtheirproductsinvitroforcommercialpurposes.However ,the rstwhole-genomesequenceforanyorganism,informationofthemostfundamentalnature,wasobtainedforthedeep-seahyperthermophileMethanococcusjannaschii(56).Althoughecologicalconsiderationsbegstudyandenzymeforagingscenariosforhyperthermophileshavenotyetbeenformulated,theacquisitionofculturablehyperther-mophilesfrommarinehydrothermalventsnowbordersonroutine.Currentrepositoriesofmarinehyperthermophiles,virtuallyallofwhichareobligatelyanaerobic,includerepre-sentativesof25genera(examplesofwhichareshowninFig.4initalics)andphysiologicalprocesses ... Inc.thermalextremeortheother,thecombinationofthisinformationwithotherbiochemicalandtheoreticalstudieshasbeenthemostrevealing(e.g.,25–27).Forexample,featuresofasuccessfulhyperthermophilicenzymecanincludeincreasedcompactness,stabiliza-tionofαhelices,increasedsaltbridgesandionpairsforstabilizingsecondarystructures,oranincreasednumberofhydrogenbonds,eachtowardretainingstabilityinthefaceofveryhighdenaturingtemperatures.Thecold-adaptedenzyme,incontrast,showsgreaterflexibilityandlesscompaction,lackssaltbridgesandionpairs,andhasareducednumberofhydrogenbonds,alltowardretainingactivityundervery-low-energynear-freezingcon-ditions.Noorganism,however,appearstohaveevolvedauniformstrategyforstabilizingorallowingactivityofallofitsenzymesatagivenextremetemperature.Instead,itssuiteofenzymesencompassesarangeofuniquecombinationsofmolecularadapationsthatreflectthehostofcomplexevolutionaryandecologicalfactors,includingacquisitionofsuccessfultraitsthroughgeneticexchangeintheenvironment(28),thatdefineacontempo-rarymicroorganism.Acommonthemeforhyperthermophilyandpsychrophily,relatingenzymesdirectlytotheproducingorganism(andthusallowingatleastsomecommonterminology),isthatthehighertheToptforgrowthoftheorganism,thehighertheToptforitsenzymes:justasenzymesoptimizedforactivityatthehighesttemperaturesclearlyderivefromhyperther-mophilesadaptedtogrowthatthehighesttemperatures(Table1),enzymeswiththelowestthermaloptimaderivefrompsychrophileswiththelowestgrowthoptima(Table2) .In fact, ... Inc.C.ForaginginSubzeroSeaIceThethreebasicfeaturesoftheenzymeforagingmodelofVetterandcoworkers(10)forparticleaggregates(Fig.1)alsopertaintotheotherendofthetemperaturespectrumformicrobiallifeandenzymaticactivityepitomizedbyseaice.Aggregatesofmineralgrainsandotherparticlesandprecipitates(includingmicroorganismsandsalts)areknowntoconcentratewithinthefluidinclusionsofseaice(6),mostnotablyintheArctic,whereseabedsedimentsentrainintocoastaliceasitforms(35).TheseaggregatesincludePOM-richdetritalparticles(36)andlargeexopolymers(37)asaresultoftheautotrophicandheterotrophiccommunitiesthatdevelopannuallywithintheicecover(38–40),aswellasgenerallyelevatedlevelsofdissolvedorganiccarbon(41,42)includingenzymes(19 ,20) .Thesea-icematrixisalsohighlyporous,especiallyinsummertime,flushingregularlywiththetidesorinfluenceofwaveswhileretainingparticleaggregatesandorganismswithinit(43,44).Evenduringwintertime(intheArctic),whensea-icetemperaturescandropbelow 20 C(Fig.2)toaslowasϪ35°C,dependingonsnowcoverandatmosphericconditions(8),interiormovementsofbrinefluidthroughfinelyconnectedchannelsarepossibleonascalerelevanttobacteriaandenzymes.Thishasbeendemonstratedbyphysicalanalysesofundisturbedicesectionsusingnuclearmagneticresonance(NMR)andtransmissionmicroscopy(45).Incontrasttoresearchonhydrothermalstructures,lessinformationisavailableontheabundanceorpossiblezonation,phylogeneticorotherwise(Fig.2),ofmicroorganismsinthesecoldestofwintertimesea-icehabitats(e.g.,18,36).Onlyin1999wasanonde-structive(nonwarming,nonmelting)methodforstudyingmicrobiallifeinsupercooledicedeveloped(36).Althoughextremetemperaturesdeterminethesolidphaseofbothhydrothermalstructures(bycontrollingmineralprecipitationreactions)andseaice(byfreezingwater),onlythehydrothermalstructureremainsintactforreadystudyattempera-tureslessextremethanthoseinsitu.Sea-icestructurechangesnonuniformlywitheveryincrementalchange(upordown)intemperature,presentingspecialchallengestoapostsamplingevaluationofinsitumicrobialcommunities,products,orprocesses.Nevertheless,thepredictionfromthethreebasicfeatures(abundantattachmentsites,organicmaterial,andporosity)thatenzymeforagingisanimportantmicrobialstrategyforgrowthandsurvivalinseaicehasbeensupportedbydirectenvironmentalmeasure-mentsinbothwintertime(18)andsummertimesea-icesamples(19 ,20) .Notonlyhavehydrolyticactivitiesonsubstrateanalogsforprotein,chitin,andvariouscarbohydratesbeenreadilydetected,but,wheremeasuredandcomparedacrossothersubzeroenviron-ments(Arcticseawaterandsinkingaggregates),thelowestthermaloptimaforenzymeactivitieswereobservedinmultiyearseaice(19).Theoptimawereconsistentlypsychro-philic,downto10°C,comparedtopreviousreportsof30°C–50°C(19 ,20, andcitationstherein)(Table2).Inotherwords,theicecoverovertheArcticOcean,whichinsomeareaspersiststhroughadecadeofwinters(rarelyifeverthecaseinAntarcticwaters),clearlyselectsforcold-adaptedandevenstrictlypsychrophilicenzymes,asitdoesforpsychrophilicorganisms(discussedlater),makingitanobviousenvironmentforcontinuedsearchanddiscoveryofnewenzymesinthisthermalclass.Specialfeaturestoconsiderinasearchforcold-adaptedenzymesinseaiceresemblethoseforseafloorsulfidestructures,albeitatsubzerotemperatures:sharpthermalgradientsinwintertimeice(Fig.2),linkedsalinity(andotherchemical)gradients(Fig.2),andthe in uence...

... alter the physicalstate or the location of the contaminants. In contrast, biodegradation is the primary processinvolved in the transformation and mineralization of xenobiotic compounds and, in the latter ... of the Copyright © 200 2 Marcel Dekker, Inc.affectthedynamicsoftheacclimationresponse,includingchemicalstructureandconcen-trationofthepollutant,presenceoforganicandinorganicnutrients,typeandphysiologicalstateofthecommunity,physical/chemicalparametersoftheenvironment(temperature,pH,redoxpotential,salinity),andbiologicalfactorssuchaspredationandcompetition(104–109).TheresponsedepictedinFig.4isacommunitylevelresponse,composedofnumerous ... results in the elimination of the pollutant and its metabolites. Abiotic degra-dation may occur, but it is less common and often results in incomplete decontamination and sometimes the formation...

... proposed in 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 ... 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.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, ... resulting low-molecular-weight products are then transported across the cell mem-brane and utilized inside the cytoplasm. The hydrolysis of polymers is an acknowledged rate-limiting step in the utilizationof...

... 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 ... © 200 2 Marcel Dekker, 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 ... 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...

... 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 ... Radosevich, SJ Traina, OH Tuovinen. Atrazine mineralization in laboratory-aged soilmicrocosms inoculated with S-triazine-degrading bacteria. J Environ Qual 26 :206 –214,1997.99. R Rai, RP Singh. Effect...