7Microbial Enzymes in the Biocontrol of Plant Pathogens and aEnzymes in the Environment: Activity, Ecology and Applications - Chapter 7PestsLeonid Chernin and Ilan ChetThe ppt

... Inc.causediseasesanddamageinagriculturalcrops.Chitinasesalsoplayanimportantphysio-logicalandecologicalroleinecosystemsasrecyclersofchitin,bygeneratingCandNsources.Someproducersofchitinases,includingTrichodermaspp.,arealsosourcesofmycolyticenzymepreparations(51,59,60).ChitinolyticenzymesaredefinedasenzymesthatcleaveabondbetweentheC1andC4oftwoconsecutiveGlcNAcunits.Onthebasisofaminoacidsequencesimilarities,allchitinaseshavebeengroupedintofamilies18,19 ,and2 0,underthemainclassofglycosylhydrolases.Mostofthemicrobialchitinasesbelongtofamily18(61,62).Evenwithinthesamefamily,chitinasesshowwidelydifferingpropertieswithrespecttosub-stratespecificity,reactionspecificity,andpHoptimum.Thechitinolyticenzymesaredi-videdintothreeprincipaltypesdependingontheiractiononchitinsubstrates.AccordingtothenomenclaturesuggestedbySahaiandManocha(59),endochitinases(EC3.2.1.14)aredefinedasenzymescatalyzingtherandomhydrolysisof1, 4- linkagesofGlcNAcatinternalsitesovertheentirelengthofthechitinmicrofibril.Theproductsofthereactionaresoluble,low-molecular-massmultimersofGlcNAcsuchaschitotetraose,chitotriose,anddiacetylchitobiose.Exochitinases,alsotermedchitobiosidasesorchitin-1, 4- -chito-biosidases(63),catalyzetheprogressivereleaseofdiacetylchitobioseunitsinastepwisefashionasthesoleproductfromthechitinchains,suchthatnomonosaccharidesoroligo-saccharidesareformed.Thethirdtypeofchitinolyticenzymeischitobiasealsotermedashexosaminidase(EC3.2.1.52)orN-acetyl- -1 ,4-d-glucosaminidase(EC3.2.1.30)belongstofamily20andalsoactsinexosplittingmodeondiacetylchitobioseandhigheranalogsofchitin,includingchitotrioseandchitotetraose,toproduceGlcNAcmonomers.RapidandspecificmethodshavebeendevelopedfordetectionandquantitativeassaysofN-acetyl-β-gluco-saminidase,chitobiosidase,andendochitinaseinsolutionsusingp-nitrophenyl-N-acetyl-β-d-glucosaminide,p-nitrophenyl-β-d-N,N′-diacetylchitotriose,andp-nitrophenyl-β-d-N,N′,N″-triacetylchitotrioseorcolloidalchitinassubstrates,respectively(64).Proceduresalsoaredescribedforthedirectassayofthesethreeenzymesaftertheirseparationbysodiumdodecylsulfate(SDS)-polyacrylamidegelelectrophoresis(PAGE)inwhichtheenzymesarevisualizedasfluorescentbandsbyusinganagaroseoverlaycontaining 4- methyl-umbelliferylderivativesofN-acetyl-β-d-glucosaminide,β-d-N,N′-diacetyl-chitobioside,orβ-d-N,N,N″-triacetylchitotriose,respectively(65).AsetofchitinolyticenzymessecretedbyvariousstrainsofT.harzianum(e.g.,TM,T-Y,39.1,CECT2413,P1ϭT.atroviride),whengrownonchitinasthesoleCsource,consistsofN-acetylglucosaminidases,endochitinases,andexochitinases(chitobiosidases).Intotal,10separatedchitinolyticenzymeswerelistedbyLorito(50);onlyonestepinthemicroparasiticprocessofT.harzianum,whichisthedissolutionofthecellwallofthetargetfungusbyenzymeactivity,mayinvolvemorethan20separategenesandgeneproductssynergisticonetoanother(Table1).TwoN-acetylglucosaminidaseswithappar-ent ... Inc.causediseasesanddamageinagriculturalcrops.Chitinasesalsoplayanimportantphysio-logicalandecologicalroleinecosystemsasrecyclersofchitin,bygeneratingCandNsources.Someproducersofchitinases,includingTrichodermaspp.,arealsosourcesofmycolyticenzymepreparations(51,59,60).ChitinolyticenzymesaredefinedasenzymesthatcleaveabondbetweentheC1andC4oftwoconsecutiveGlcNAcunits.Onthebasisofaminoacidsequencesimilarities,allchitinaseshavebeengroupedintofamilies18,19 ,and2 0,underthemainclassofglycosylhydrolases.Mostofthemicrobialchitinasesbelongtofamily18(61,62).Evenwithinthesamefamily,chitinasesshowwidelydifferingpropertieswithrespecttosub-stratespecificity,reactionspecificity,andpHoptimum.Thechitinolyticenzymesaredi-videdintothreeprincipaltypesdependingontheiractiononchitinsubstrates.AccordingtothenomenclaturesuggestedbySahaiandManocha(59),endochitinases(EC3.2.1.14)aredefinedasenzymescatalyzingtherandomhydrolysisof1, 4- linkagesofGlcNAcatinternalsitesovertheentirelengthofthechitinmicrofibril.Theproductsofthereactionaresoluble,low-molecular-massmultimersofGlcNAcsuchaschitotetraose,chitotriose,anddiacetylchitobiose.Exochitinases,alsotermedchitobiosidasesorchitin-1, 4- -chito-biosidases(63),catalyzetheprogressivereleaseofdiacetylchitobioseunitsinastepwisefashionasthesoleproductfromthechitinchains,suchthatnomonosaccharidesoroligo-saccharidesareformed.Thethirdtypeofchitinolyticenzymeischitobiasealsotermedashexosaminidase(EC3.2.1.52)orN-acetyl- -1 ,4-d-glucosaminidase(EC3.2.1.30)belongstofamily20andalsoactsinexosplittingmodeondiacetylchitobioseandhigheranalogsofchitin,includingchitotrioseandchitotetraose,toproduceGlcNAcmonomers.RapidandspecificmethodshavebeendevelopedfordetectionandquantitativeassaysofN-acetyl-β-gluco-saminidase,chitobiosidase,andendochitinaseinsolutionsusingp-nitrophenyl-N-acetyl-β-d-glucosaminide,p-nitrophenyl-β-d-N,N′-diacetylchitotriose,andp-nitrophenyl-β-d-N,N′,N″-triacetylchitotrioseorcolloidalchitinassubstrates,respectively(64).Proceduresalsoaredescribedforthedirectassayofthesethreeenzymesaftertheirseparationbysodiumdodecylsulfate(SDS)-polyacrylamidegelelectrophoresis(PAGE)inwhichtheenzymesarevisualizedasfluorescentbandsbyusinganagaroseoverlaycontaining 4- methyl-umbelliferylderivativesofN-acetyl-β-d-glucosaminide,β-d-N,N′-diacetyl-chitobioside,orβ-d-N,N,N″-triacetylchitotriose,respectively(65).AsetofchitinolyticenzymessecretedbyvariousstrainsofT.harzianum(e.g.,TM,T-Y,39.1,CECT2413,P1ϭT.atroviride),whengrownonchitinasthesoleCsource,consistsofN-acetylglucosaminidases,endochitinases,andexochitinases(chitobiosidases).Intotal,10separatedchitinolyticenzymeswerelistedbyLorito(50);onlyonestepinthemicroparasiticprocessofT.harzianum,whichisthedissolutionofthecellwallofthetargetfungusbyenzymeactivity,mayinvolvemorethan20separategenesandgeneproductssynergisticonetoanother(Table1).TwoN-acetylglucosaminidaseswithappar-ent ... involved in triggering the expression of the 42-kD endochitinase and the 73-kD N-acetyl-β-d-glucosaminidase. This last enzyme revealed high similarityto N-acetyl-glucosaminidases from other eukaryotes,...

... of PLDf,which contains a Phox (PX) and a Pleckstrin (PH)homology domain in the N-terminal region, all the other PLD isoenzymes possess a calcium- and phos-pholipid-binding domain (C2 domain) ... being dominant. The fluorescence and near-UV CD spectra point to tight packing of the aromatic residues in the core of the protein. From the near-UV CD signals and activity data as a function of ... superfamily. However, there is low similar-ity in the remaining parts of the molecules. Most mam-malian PLDs contain a PX and a PH domain instead of the C2 domain in plants, whereas microbial PLDslack...

... 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 ... Inc.Thereisthepotentialtouseanintegratedmeasurementofanumberofenzymeactivities(asdiscussedlater),inconjunctionwithotherphysical,chemical,andmicrobio-logicalmeasurements,inassessingsoilquality.Somehydrolaseactivitiesdonotshowwideseasonalvariation,probablybecauseofthelargeamountofactivityassociatedwithenzymesstabilizedbysoilcolloids.Thisprovidesagreatadvantageovermicrobiologicalmeasurementssuchasrespiration,whichvarysowidelywithinaseasonoronayear-to-yearbasisandmakeitdifficulttofindtrendsoridentifytheimpactsofdifferentmanage-mentsystems.Dehydrogenaseactivityisanintracellularprocessthatoccursineveryviablemicro-bialcellandismeasuredtodetermineoverallmicrobiologicalactivityofsoil(40,41) .The problemwiththisisthattheelectronacceptors(2,3,5-triphenyltetrazoliumchloride[TTC](seealsop.19)or2-p-Iodophenyl-3-p-nitrophenyl-5-phenyltetrazoliumchloride[INT](seealsop.13))usedintheassaysarenotveryeffective,andthusthemeasurementsmayunderestimatethetruedehydrogenaseactivity(41).Anotherpotentiallyconfusingaspectofthesestudiesarisesasaconsequenceofsoilcollectionandpretreatment.AccordingtoNannipieriandcoworkers(41),enzymeactivitymeasurementsarecarriedoutafterremovalofvisibleanimalsandplantdebrisandonsievedsoilsamplesunderlaboratoryconditions.Thusthemeasuredactivitiesofthesesamplesmaydependonthemetabolicprocessesorenzymeactivitiesassociatedonlywiththemicrobialcells.Conversely,whenratesofmetabolicprocesses,suchasrespiration,aremeasuredinthefield,thecontributionsoflivingrootsandanimalsaswellasmacro-scopicorganicdebrisarerecorded.Inotherwords,totalbiologicalratherthanmicrobiolog-icalactivitiesaremeasured(41).B.EnzymeActivities:MethodologyandInterpretationAccordingtothereviewbySkujins(40),Woodsin1899suggestedthatextracellularenzymescouldbepresentandactiveinsoil .The rstmeasurementsofenzymeactivitiesinsoilweredoneoncatalaseandperoxidaseactivitiesfrom1905to1910(40).Sincethen,theactivityofdozensofenzymeshasbeendetectedinsoil.Obviouslythenumberofenzymesisconsiderablygreaterthanthosemeasuredbecauseofthemultitudeofmicro-bial,faunal,andplantspeciesinhabitingsoil(46).Inaddition,theactivitymeasuredbymanyassayscannotbeascribedtotheactionofasingleenzyme.Thusdehydrogenaseactivityisdeterminedbythemultipleenzymereductionofasyntheticsubstrate(TTCorINT)duetoanoxidationofgenerallyunknownendogenoussubstrateswhoseconcentra-tionisalsounknown(46).Casein-hydrolyzingactivitiesaremeasuredwithoutspecificidentificationofthebondhydrolyzedorofallproductsformed.Itisimportanttoempha-sizethatevenwhenallthecomponentsofthereactionareknown,forexample,inthecaseofureaseassays,differentenzymesfromdifferentsources(microbial ,plant, oranimalcells)catalyzethesamereaction.Tables 2and3 (99–105)reportarangeofactivitiesofenzymescommonlyinvesti-gated ... aspartase-Ca-montmoril-lonite systems (159). Deamination of l- and d-glutamic and aspartic amino acids and of their 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, ... Noncompetitive inhibi-tion of the activity of exoproteases by Cu2ϩions (43) and inhibition of α-glucosidase, - glucosidase, N-acetyl-glucosaminidase, and alkaline phosphatase by H2S in natural ... Control of the Synthesis and Activity of Ectoenzymes The complex environmental regulation of ectoenzyme synthesis and activity has beendemonstrated in studies of bacterial β-glucosidase and aminopeptidase...

... 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 ... BIOTECHNOLOGICALASPECTS OF MARINE-DERIVED ENZYMES Until recently, investigations of marine-derived enzymes were hampered by the necessity of isolating a microbe in pure culture and then obtaining sufficient ... Inc.inthefreeform,andconsequentlyavailableforrapiduptake,remainsunknown.Thisadsorptionandtheconcurrentloweravailabilityforbacterialuptakemightcauseanunder-estimationoftheactualbacterialproductiononandinpolysaccharide-richmaterialsuchasmarinesnow(44),relativetobacterialenzymeactivity.ThecouplingbetweenhydrolysisanduptakeofDOMinparticle-associatedandfreebacteriaisstillnotfullyunderstood.Thereasonswhytheattachedbacteriabenefitsolittlefromtheirstronghydrolyticactivities,iftherearenolimitingfactorsinterferingwiththeuptakeofenzymatichydrolysisproducts,areunknown.Thisfundamentaldiscrepancyshouldbemorethoroughlyinvestigatedinordertoimproveunderstandingofthebiogeo-chemicalfluxoforganicmatterandtheroleofbacteriainthecyclingofDOMintheocean.Inanycase,itiswellacceptedthatparticledecomposition(45)contributessignificantlytothelossoforganicmaterialfromsettlingparticlesduringsinkingandthusdeterminestheefficiencyofthebiologicalCpump(organicmattertransportfromtheseasurfacetotheseabed).D.EnvironmentalFactorsInfluencingEnzymaticActivityThemagnitudeofthemainextracellularenzymeactivitiesinmarinewaterisfrequentlyintheorderaminopeptidaseϾphosphataseϾβ-glucosidaseϾchitobiaseϾesteraseϾα-glucosidase.However,exceptionsmayoccur,asobservedbyChristianandKarl(46)intheequatorialPacific,whereβ-glucosidasewasaboutfourtimeshigherthanaminopep-tidase.Thissuggeststhattheremaybefactorsregulatingactivitiesonalargescale.How-ever,knowledgeofglobalregulatingfactorsisscarce.ChristianandKarl(47)foundthathistidineandphenylalanineinhibitedaminopeptidaseexpressioninAntarcticwaters.Like-wise,KimandLipscomb(48)suggestedthatmetalsmayberegulatingfactorsforproteases(leucineaminopeptidaseseemstobeprincipallyaZn2ϩ-dependentenzyme).ThiswasespeciallyduetoZn2ϩ(whichisrareinmarinewaters),butMn2ϩ,Co2ϩ,Fe2ϩ,andMg2ϩmightalsoplayarole(47–50).Inthesurfacelayeroftheocean,ultraviolet-Bradiationcanbeimportant,mainlythroughphotochemicaldegradationoftheextracellularenzymes(51,52).Withrespecttophosphataseactivity,theabundanceofinorganicPisregardedasaregulatingfactor,particularlyfortheP-limitedregionsintheoceans(53–55).However,dissolvedorganicphosphorus(DOP)andparticulateorganicPalsoshouldbeconsidered(56).Furthermore,mechanismsofphosphataseregulationaredifferentforbacteriaandphytoplankton.WhilethephosphatasesofphytoplanktonseemtoberegulatedstrictlybyinorganicPconcentrations(49,57–59),thismechanismisnotsoclearforbacterialphosphatases.ThelattermaytargetCandNratherthanPsupply,aspointedoutforthelimneticenvironmentbySiudaandGu¨de(60)andforthedeepandC-limited,butphos-phate-replete,oceanbyHoppeandUllrich(61).Inanycase,regardlessofenvironmentalfactors,variationofspeciescompositionwithinthebacterialcommunitycansignificantly in uencethedistributionofenzymeactivitiesinthesea(62,63).Theeffectsofenvironmentalfactorsonenzymeregulationarereflectedbythediver-sityofextracellularenzymes,asexpressedinthepossiblerangesofKmandthepatternsofindividualcell-specificenzymepotentials(Table2,Table3).InformationontheKmvalues...

... 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 ... with the GMM inocu-lum, in the presence of kanamycin, showed the only impact of the GMM compared tothat the wild-type inoculum. The shift toward K strategists (i.e., slower-growing organ-isms)...

... 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,themodulation of ... 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 ... some of the important enzymes and the role of these enzymes in the penetration of the fungus inside the plant root are discussed later.A. CellulasesCellulose is the best known of all plant...

... 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 ... Inc.(nitrificationanddenitrificationeffects)aswellasbyprotectionandcreationofwetlands(4,7,39,57,85,122).Itisagainstthisbackdropofthemajorenvironmentalrelevanceoftheenzymesofnitrogenandcarboncyclingprocessesthatthischapterispresented.Theutilityofsoilenzymeactivitiesasindicatorsofsoilqualityandinmonitoringoftheeffectsofsoilpollutionispresentedelsewhere(14,34,60,116,131)andinChapters15,16 ,and1 7 .The general ... 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 of soil...

... 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 ... that the prominence of ligninase-producing basidiomycetes (35) in terrestrial systems and pectinase-producing hyphomy-cetes (7,89) in aquatic systems probably affects the functional profile and ... 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...

... one-yearperiod together with the cellulase- and lignin-degrading ability of the fungi. The firstcolonizers were Mucor and Cladosporium spp. The number of Mucor spp. isolates de-creased after the ... 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 ... been introduced over the years; these include vanilin, indulin, ferrulic acid, and, most importantly,14C-labeled synthetic lignins. Various fungal enzymes are involved in lignin degradation, including...