Enzymes in the Environment: Activity, Ecology and Applications - Chapter 5 ppt

... 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.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 ... 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.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 ... 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(1 45, 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)wasabove 55( 150 )(Fig.14).Moreover, ... 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...

... indicating that the ester probably interfered with nutrient cycling’’(103). In the former study, urease and acid- and alkaline-phosphatase activities were tem-porarily reduced, whereas in the ... c1 and c2, ED 50 (1), and ED 50 (2)representuninhibited rates and ED 50 values for the full- and partial-inhibition models, respectively; c2a/b,minimum (asymptote) for the partial-inhibition ... Although the reason for the choice of these particular enzymes is not clear, a dis-criminant function combining seven properties, including alkaline phosphatase and argi-nine deaminase activities,...

... 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 ... 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 ... 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...

... 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 ... 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.Althoughthisstudyinvolvedtheuseofageneticallymodifiedmicrobe,themodi - cationswerenotintendedtohaveafunctionalimpact;theywereinsertedasgeneticmark-ers.Asecondstudycomparingtheeffectofthesamegeneticallymarkedstraintothatofafunctionallymodifiedstrainshowedeffectsthataremoreinteresting(36).Theaimofthisworkwastodeterminetheimpactintherhizosphereofwildtypealongwithfunction-allyandnonfunctionallymodifiedPseudomonasfluorescensstrains.Thewild-typeF113straincarriedageneencodingtheproductionoftheantibiotic2,4-diacetylphloroglucinol(DAPG),usefulinplantdiseasecontrol,andwasmarkedwithalacZYgenecassette .The firstmodifiedstrainwasafunctionalmodificationofstrainF113withrepressedproductionofDAPG,creatingtheDAPGnegativestrainF113G22.Thesecondpairedcomparisonwasanonfunctionalmodificationofwild-type(unmarked)strainSBW 25, constructedtocarrymarkergenesonly,creatingstrainSBW25EeZY-6KX.Significantperturbationswererecordedintheindigenousbacterialpopulationstruc-ture;theF113(DAPGϩ)straincausedashifttowardslower-growingcolonies(Kstrate-gists)comparedwiththenon-antibiotic-producingderivative(F113G22)andSBW 25 strains.TheDAPGϩstrainalsosignificantlyreduced,incomparisonwiththoseoftheotherinocula,thetotalPseudomonassp.populations,butdidnotaffectthetotalmicrobialpopulations.ThesurvivalofF113andF113G22wasanorderofmagnitudelowerthanthatoftheSBW25strains.TheDAPGϩstraincausedasignificantdecreaseintheshoot-to-rootratioincomparisontothatofthecontrolandotherinoculants,indicatingplantstress.F113increasedsoilalkalinephosphatase,phosphodiesterase,andarylsulfataseac-tivities(Table2)comparedtothoseofthecontrols.Theotherinoculareducedthesameenzyme ... 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 ... causinga shift toward slower-growing organisms. The community structure with the GMM inocu-lum, in the presence of kanamycin, showed the only impact of the GMM compared tothat the wild-type inoculum....

... 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 ... 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.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 ... Microbial Enzymes in Aquatic Environments. New York: Springer-Verlag, 1991, pp 25 59 .10. JS Clein, JP Schimel. Reduction in microbial activity in birch litter due to drying and rewettingevents. ... amendment varied with the lignin content of the litter. Dogwood,a fast-decomposing, low-lignin litter, decomposed up to 25% faster than did the controlplots. Maple, intermediate in lignin content, decomposed...

... 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. ... sterilefreshleaves,cellulose,lignin,andphenoldegradationwasinitiatedimmediately.Fourteendayslater,when20%ofthecellulosehadbeendegraded,therateoflignindegradationincreased.Decompositionwasrapidduringthefirstmonthbutvirtuallyceasedafterfourmonths.Lowresourcequalityandadverseenvironmentalconditions(e.g.,lowwateravail-ability)resultinlowdecompositionrates.ThishasbeenexaminedbyincubatingpineneedlesinlitterbagsinasouthernItalianpineforest(49).BoththeC/Nratioandthelignincontentofthelitterwerehigh.MeasurementofbiologicalparameterssuchasCO2evolutionandfungalbiomassoverathree-yearperiodrevealedasignificantpositivecorre-lationbetweenrespirationrateandmoisturecontentofthelitter.Therewasnoobviousrelationshipbetweenfungalbiomassandothermeasuredparameters(i.e.,littermassloss,lignincontent,andnitrogencontent).Itwasconcludedthatsincethelitterwasverydryformostoftheyear,anautochthonousfungalflorahaddevelopedthatwasabletodegradetheselittertypesunderadverseconditionsalbeitatalowrate.Theexamplesofinteractionsbetweensubstratesandfungalgroupsmentionedandtheinfluenceofdifferentconcentrationsofsubstratesillustratethecomplexanddynamicprocessesinvolvedinlitterdecomposition.Inthenextsectionthesuccessionalstagesofdecompositionarediscussedinthecontextofenzymeactivity.IV.FUNGALPOPULATIONSANDENZYMEACTIVITYNumerousstudiesonfungalsuccessionhavebeenpublished,manyofwhichdiscusstheidentificationoffungiatdifferentstagesofdecomposition(1).However,theemphasisisusuallyontaxonomicidentityratherthanonenzymaticdiversity.Thosegeneramostfre-quentlymentionedinconnectionwithearlycolonizationoftheorganicdebrisinthetem-peratezoneareAlternaria,Aureobasidium,Cladosporium,andEpicoccum.Inherreview,Frankland(1)concluded,‘‘Letusecologistsnotneglecttostudyingreaterdepthmoreofthestarperformersinfungalsuccession,onwhichthemaintenanceofentireecosystemsmaydepend.’’Inthiscontext‘‘starperformers’’encompasstheimportantenzymeproduc-ersandhencethekeydecomposers.ThelinkbetweentaxonomicandfunctionaldiversityinthefungalpopulationhasbeendiscussedinreviewsbyMiller (50 )andZakandVisser (51 ),bothofwhichemphasizetheimportanceofsuccessionstudies.Therelationshipbetweenfungalsuccessionandtheenzymaticpotentialofthefungihasbeenobservedduringdecompositionofforestlitter,e.g.,ofalder(2 ,52 57 )andbeech (54 57 )(seeTable2).Onbeechleaves,fungalspeciesofthegeneraAureobasidium,Cladosporium,Epi-coccum,andAlternariaappearfirst,althoughMucor,Phoma,andAcremoniumareoftenearlycolonizers(Table3).Acremoniumspp.isolatesattackcelluloseandchitinaswellas...

... 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 ... the α-chymotrypsin catalytic site. This is due to an interaction involving positively chargedHisϩ -4 0 and Hisϩ -5 7 imidazole and Alaϩ -1 49 (alanine) end chain aminium that control the initial specific...