3 Ecological Significance of Bacterial Enzymes in the Marine Environment Hans-Georg Hoppe Institute of Marine Science, Kiel, Germany Carol Arnosti University of North Carolina, Chapel Hill, North Carolina Gerhard F. Herndl Netherlands Institute of Sea Research (NIOZ), Den Burg, The Netherlands I. INTRODUCTION The general biochemical role of extracellular enzymes in the sea is similar to that in other aquatic environments. However, in order to understand the ecological significance of enzymes in the sea specific marine environmental factors have to be considered, e.g., a) seawater is a highly diluted medium interspersed with hot spots of organic matter con- centration, aggregation, and decomposition (1). b) as a result of hydrographic conditions, the oceans are characterized by distinct horizontal and vertical zonations. The deep sea, in particular, with its enormous volume, depends entirely on substrate supply from sinking material produced in the surface layer (2). c) in the sea, low-molecular-weight organic matter that persists as dissolved organic carbon (DOC) is less bioreactive and more strongly diagenetically altered than the bulk of high-molecular-weight matter (3,4). d) the deep seabed receives very little of the total surface-derived primary productivity, and much of this organic matter is strongly altered. Finally, at chemical and hydrographic discontinuities in the sea (e.g., fronts, boundary layers, and oxygen, nutrient, and salinity gradients), drastic changes occur in microbial species diversity and enzymatic properties. Organic material, prone to bacterial degradation on nongeological time scales, is actively involved in biogeochemical cycles. Such material originates from phytoplankton primary production and from ‘sloppy’ feeding of zooplankton as well as from the excre- tions of all kinds of organisms. Currently, the spectrum of extracellular enzymes investi- gated in the sea is relatively limited, comprising principally hydrolytic enzymes such as proteases, glucosidases, chitinase, lipase, and phosphatase. A larger variety has been inves- tigated in limnetic systems (5). The principal focus of this chapter is on the ecological significance of extracellular enzymes in marine waters and sediments ranging from microscales to oceanwide scales. Copyright © 2002 Marcel Dekker, Inc. Investigationsofextracellularenzymesfrommarineanimalsandenzymesisolatedfrom prokaryotesareconsideredonlyifaclearconnectiontomarineecologyisestablished. Thetermextracellularenzymesisusedthroughoutthischapter,whereasChro ´ st(5)distin- guishesbetweenectoenzymesandextracellularenzymes.Ectoenzymesaredefinedby Chro ´ st(5)andinChapter2asenzymeslocatedintheperiplasmicspaceorattachedto theoutermembraneofthebacterialcell.Extracellularenzymesareenzymesfreelydis- solvedinthewaterorattachedtoparticlesotherthantheenzyme-synthesizingcell.In thischapter,however,thetermextracellularenzymesreferstobothectoenzymesand extracellularenzymes,unlessotherwisestated. Earlystudiesonthefateoforganicaggregatesanddissolvedpolymersinthesea werepresentedbyRiley(6),Walsh(7),andKhailovandFinenko(8).Overbeck(9)re- viewedtheearlystudiesonextracellularenzymeactivityintheaquaticenvironment. II.ECOLOGICALPRINCIPLESOFENZYMATICPATTERNS INTHESEA A.TheConceptoftheMicrobialLoopandtheRoleofExtracellular Enzymes Themicrobialloop(10)encompassesthecombinedactivitiesofautotrophicandheterotro- phic—eukaryoticaswellasprokaryotic—organismssmallerthan20µm.Theseorgan- isms,representedbybacteria,nanoflagellates,ciliates,andphototrophicprochlorophytes, aswellascyanobacteria,formafoodweboftheirown,looselyconnectedtothefood webofthelargergrazers.Ingeneral,thenutritionalbasisofthemicrobialfoodwebis providedbythepoolofdissolvedorganicmatter(DOM)andparticulateorganicmatter (POM).TheDOMpoolisapriorireservedforbacterialutilization,whereascompetition withmetazoansoccursforPOM.Thiscompetitionisdeterminedbythebacterialpotential forenzymaticdissolutionofPOMontheonehandandthefeedingactivityofthemetazo- ansontheotherhand.Thebulkofboththedissolvedandparticulateresources,however, requiresenzymatichydrolysispriortouptakebybacteria(Fig.1).Thustheenzymatic activitiesofbacteriainitiateorganiccarbon(C)remineralizationanddefinethetypeand quantityofsubstrateavailabletothetotalmicrobialfoodweband,tocertainextent,also tothetoppredatorsinthesystem. B.FreeandAttachedEnzymeActivity Generally,extracellularenzymesmaybeboundtothecell(definedasectoenzymesby Chro ´ st[5])orinthefreeandadsorbedstate(11,12).Mostofthetotalenzymeactivity inseawaterhasbeenfoundtobeassociatedwiththeparticlesizeclassdominatedby bacteria(Ͼ0.2µm–3µm)(13,14)(Table1).Dissolvedenzymes(15)andlargeparticles Ͼ8 µm generally contribute only minor parts to the total enzyme activity. In estuaries, however, which are characterized by strong gradients and fluctuations of turbidity and salinity, total enzyme activity can be dominated by particle size classes Ͼ3 µm. Enzyme activity measured in such particles originated mainly from attached bacteria, leading to the conclusion that particle-attached bacteria accounted for most of POM degradation in these estuaries (16). Another example of the dominance of particle-associated enzyme activity is marine snow. Although bacterial production was not enhanced on snow parti- cles, enzyme activities (α- and β-glucosidases, leucine amino peptidase) of marine snow– Copyright © 2002 Marcel Dekker, Inc. Figure1Extracellularenzymeactivity(EEA),theinitialstepofthemicrobialloop.Theenzy- maticconversionofparticulateorganicmatterandmacromolecularexudatestodissolvedorganic matter(DOM)triggersthemicrobialloop.Arrowsindicatethepathwaysofdegradation,grazing, andpredation. attachedbacteriaweresignificantlyhigherthanthoseoffree-livingbacteria,intermsof bothabsoluteandper-cellrates(17).Similarobservations,withrespecttotherelationship betweenenzymeactivitiesandaminoacidincorporationofparticle-associatedbacteriain theSanFranciscoBay,werereportedbyMurrell,etal.(18).Theenzymeactivitiesof bacteriaassociatedwiththerecentlyexploredtransparentexopolymerparticles(TEPs)- whichcanharbor2%to25%oftotalbacteriainthesea(19,20)-havenotyetbeenexam- ined. Significantdifferencesintheextracellularenzymeactivitypercellbetweenparticle- attachedandfree-livingbacteriafrequentlyhavebeenreportedalthoughthesedifferences arenotalwaysobservedandmostlikelydependonthequalityandcompositionofthe particlesaswellasonthenatureofthecolonizingbacteria(17,21–26)(Table2).Metaboli- cally active particle-attached bacteria commonly have a larger polysaccharidic capsule than do free-living bacteria (27). Because the majority of extracellular enzymes are embed- ded in the capsular envelope of metabolically active bacteria, a larger capsule potentially could harbor a greater quantity of enzymes. It also has been observed that the capsular Copyright © 2002 Marcel Dekker, Inc. Table 1 Particle-Attached and Free (Ͻ0.2-µm) Extracellular Enzyme Activity of Different Enzymes in Different Habitats Percentage Percentage Percentage Enzyme, Size of total Size of total Size of total Environment Conditions substrate class (µm) activity class (µm) activity class (µm) activity Reference Estuary Salinity- leu-AMP Ͻ3 ϳ8–23 Ͼ3 ϳ77–92 (16) turbidity β-glucosidase Ͻ3 ϳ5toϳ50 Ͼ3 ϳ50 to ϳ95 gradient Northern Zooplankton leu-AMP Ͻ0.2 60–86 (29) Adriatic enzyme re- α-glucosidase 4–11 Sea lease β-glucosidase 0.6–10 California Surface sea leu-AMP Ͻ0.2 0.2 Ͻ140–80Ͼ1 ϳ20–60 (37) Bight water North Sea leu-AMP Ͻ0.2 0–30 (15) Santa Monica Above 100 m leu-AMP Ͻ0.2 Ͻ30 0.2–0.8 70–75 (14) Basin San Francisco Spring and leu-AMP Ͼ1 47–76 av. 65 (18) Bay summer β-glucosidase Ͼ1 15–87 av. 56 Kiel Fjord, Mesotrophic phosphatase Ͻ0.2 33 0.2–3 14 3–150 53 (219) chitobiase Ͻ0.2 4 0.2–3 96 3–150 0 Northern Red Oligotrophic phosphatase 0.2–2 50–71 2–20 12–27 Ͼ20 3–37 (77) Sea Copyright © 2002 Marcel Dekker, Inc. Table 2 Specific Extracellular Enzyme Activity per Bacterial Cell (amol cell Ϫ1 h Ϫ1 ) of Enzymes in Different Habitats Environment Conditions leu-AMP Lipase P-ase α-Glucosidase β-Glucosidase Chitobiase Reference Trophic gradient Eutrophic 31.6 10.3 1.69 0.18 (113) Mesotrophic 8.1–9.3 Oligotrophic 75.6 35.1 0.4 0.06 Santa Monica Oligotrophic ϳ78–618 (14) Basin Adriatic Sea Marine snow 432–4996 7–40 6–140 (17) Selected aggregates Experimental av. 242 Ϯ 493 (40) Seawater av. 52.5 Ϯ 15 California Bight 44 Isolates from 4–3810 0.2–584 0.7–410 0–8 0–35 0–559 (148) marine sources Baltic Sea Tank incubations 2–14 (220) Baltic Sea Summer 0.3–5 0.1–3.3 0.2–3.7 0.7–3.3 (115) Autumn 20–237 0.1–1 0.2–2 0.5–5.8 Arabian Sea Euphotic zone 6.6–23.2 0.4–3.6 0.16–0.22 (61) Deep water 33–118 5.6–23.4 0.27–1.18 Oman coast, upwel- Euphotic zone 12.6–46.9 1.2–8.3 0.02–1.2 (61) ling Deep water 455–1817 10.8–86.2 7.7–52.5 Coastal lagoon Hypertrophic LT 218–478 6.9–25.0 (221) HT 188–625 5.4–12.9 San Francisco Bay Cells Ͻ1 µm 7.2–12 0.16–0.57 (18) All cells 16–31 0.47–1.60 Uranouchi Inlet, Surface water 23.2–1017 (114) Japan Bottom water 21.1–270 LT, low tide; HT, high tide. Copyright © 2002 Marcel Dekker, Inc. layeriscontinuouslyrenewedbythebacteria(28).Metabolicallyinactivebacteria,in contrast,areusuallydevoidofacapsule(27),andalargerfractionofactivebacteriahas beenfoundinmarinesnowthaninfree-livingbacteria(27).Consequently,ahigherpro- portionofactive,particle-associatedbacteriamightresultinanoverallhigherextracellular enzymeactivitypercell. Thepooloffreedissolvedenzymes(i.e.,enzymesthatpassthrough0.2-µm-pore- sizefilters)isfueledbyvarioussources.Inadditiontoenzymesreleasedbybacteria,they canbederivedfromprotozoasuchasflagellatesandciliatesandfrommesozooplankton (e.g.chitinase).Obviously,duringperiodsofhighzooplanktongrazingactivity,selected enzymescancontributethemajorityofthebulkactivity(29)(Table1),butthisisnota common feature. Special patterns of distribution were recorded for phosphatases (13,30,31), which are generated not only by bacteria but also by phytoplankton, cyanobac- teria (32,33), and macroalgae (34). C. The Particle Decomposition Paradox and the Biological C Pump Organic particles represent the nutritional basis for bacteria, and life in general, in the aphotic zone of the marine environment. However, microscopic analysis has revealed that particles are frequently less heavily colonized by bacteria than expected. Nevertheless, below the euphotic zone, particle decomposition has to supply the entire microbial commu- nity including the free-living bacteria. A key to understanding this paradox lies in the enhanced individual enzyme activity of the attached bacteria (18,21,35,36) and probably also in the extracellular release of endo-enzymes by these bacteria (37,38). By hydrolyzing macromolecular linkages in an endo- fashion (i.e., hydrolyzing the nonterminal linkages in a polymer), these enzymes are able to break up the complex polymers inside the parti- cles. Both processes potentially create a surplus of dissolved monomeric or oligomeric hydrolysis products from the particles that are not entirely taken up by the attached bacteria (loose hydrolysis-uptake coupling). Escaping into the surrounding water, these substrates support the nutrition of free-living bacteria (39,40). A loose hydrolysis-uptake coupling frequently has been reported for particle-attached bacteria, whereas tight coupling has been reported between hydrolysis of DOM and uptake of the resulting monomers (17,22,40). Other studies, however, have not revealed a difference in the hydrolysis-uptake cou- pling between attached and free-living bacteria (24,36). In two recently published investi- gations on the extracellular enzyme activity of marine snow–associated bacteria, no evi- dence was found that glucosidase and aminopeptidase activity in marine snow–associated bacteria were less tightly coupled to the uptake of the respective monomers than in free- living bacteria (23,26). Furthermore, in a number of studies using thymidine and leucine incorporation into bacterial deoxyribonucleic acid (DNA) and protein (41,42), respec- tively, as an estimate of bacterial C production, growth and the hydrolytic activities of attached and free-living bacteria were compared (17,24,36,40). On the basis of such bacte- rial production estimates and concurrently measured hydrolytic activity, it was concluded that the C demand of attached bacteria was lower than the amount of C cleaved by enzy- matic activity, hence indicating a loose hydrolysis-uptake coupling (43). However, it is well known that leucine and especially thymidine are efficiently adsorbed to polysaccha- rides. This adsorbed (radiolabeled) thymidine and leucine is taken up at significantly lower rates than their nonadsorbed, truly dissolved counterparts (44). Since determinations of the saturating substrate concentrations are rarely made in such investigations (for logistic reasons), the amount of radiolabeled thymidine and leucine actually available for bacteria Copyright © 2002 Marcel Dekker, Inc. inthefreeform,andconsequentlyavailableforrapiduptake,remainsunknown.This adsorptionandtheconcurrentloweravailabilityforbacterialuptakemightcauseanunder- estimationoftheactualbacterialproductiononandinpolysaccharide-richmaterialsuch asmarinesnow(44),relativetobacterialenzymeactivity. ThecouplingbetweenhydrolysisanduptakeofDOMinparticle-associatedandfree bacteriaisstillnotfullyunderstood.Thereasonswhytheattachedbacteriabenefitsolittle fromtheirstronghydrolyticactivities,iftherearenolimitingfactorsinterferingwiththe uptakeofenzymatichydrolysisproducts,areunknown.Thisfundamentaldiscrepancy shouldbemorethoroughlyinvestigatedinordertoimproveunderstandingofthebiogeo- chemicalfluxoforganicmatterandtheroleofbacteriainthecyclingofDOMintheocean. Inanycase,itiswellacceptedthatparticledecomposition(45)contributessignificantlyto thelossoforganicmaterialfromsettlingparticlesduringsinkingandthusdeterminesthe efficiencyofthebiologicalCpump(organicmattertransportfromtheseasurfacetothe seabed). D.EnvironmentalFactorsInfluencingEnzymaticActivity Themagnitudeofthemainextracellularenzymeactivitiesinmarinewaterisfrequently intheorderaminopeptidaseϾphosphataseϾβ-glucosidaseϾchitobiaseϾesteraseϾ α-glucosidase.However,exceptionsmayoccur,asobservedbyChristianandKarl(46) intheequatorialPacific,whereβ-glucosidasewasaboutfourtimeshigherthanaminopep- tidase.Thissuggeststhattheremaybefactorsregulatingactivitiesonalargescale.How- ever,knowledgeofglobalregulatingfactorsisscarce.ChristianandKarl(47)foundthat histidineandphenylalanineinhibitedaminopeptidaseexpressioninAntarcticwaters.Like- wise,KimandLipscomb(48)suggestedthatmetalsmayberegulatingfactorsforproteases (leucineaminopeptidaseseemstobeprincipallyaZn 2ϩ -dependentenzyme).Thiswas especiallyduetoZn 2ϩ (whichisrareinmarinewaters),butMn 2ϩ ,Co 2ϩ ,Fe 2ϩ ,andMg 2ϩ mightalsoplayarole(47–50).Inthesurfacelayeroftheocean,ultraviolet-Bradiation canbeimportant,mainlythroughphotochemicaldegradationoftheextracellularenzymes (51,52).Withrespecttophosphataseactivity,theabundanceofinorganicPisregardedas aregulatingfactor,particularlyfortheP-limitedregionsintheoceans(53–55).However, dissolvedorganicphosphorus(DOP)andparticulateorganicPalsoshouldbeconsidered (56).Furthermore,mechanismsofphosphataseregulationaredifferentforbacteriaand phytoplankton.Whilethephosphatasesofphytoplanktonseemtoberegulatedstrictly byinorganicPconcentrations(49,57–59),thismechanismisnotsoclearforbacterial phosphatases.ThelattermaytargetCandNratherthanPsupply,aspointedoutforthe limneticenvironmentbySiudaandGu ¨ de(60)andforthedeepandC-limited,butphos- phate-replete,oceanbyHoppeandUllrich(61).Inanycase,regardlessofenvironmental factors,variationofspeciescompositionwithinthebacterialcommunitycansignificantly influencethedistributionofenzymeactivitiesinthesea(62,63). Theeffectsofenvironmentalfactorsonenzymeregulationarereflectedbythediver- sityofextracellularenzymes,asexpressedinthepossiblerangesofK m andthepatterns ofindividualcell-specificenzymepotentials(Table2,Table3).InformationontheK m values of marine bacteria, however, is scarce. Proteinase affinities seem to be higher in oligotrophic than in eutrophic regions. K m values observed in Antarctic regions at in situ temperature were similar to those in warmer regions; the relationship does not seem to hold for Arctic environments (Table 3). Cell-specific enzyme activities vary over a wide range. They are low in eutrophic waters, but relatively high in oligotrophic waters and Copyright © 2002 Marcel Dekker, Inc. Table3K m Values(Apparent)ofExtracellularEnzymesinDifferentMarineHabitats K m EnvironmentConditionsEnzyme,substrate(µmolL Ϫ1 )Reference TrophicgradientEutrophicleu-AMP47.6(113) Oligotrophic0.71 AntarcticaϪ1.7°Ctoϩ2°Cleu-AMP67–132(47) Ϫ1.7°Ctoϩ20°C48–218 MesocomsBeforestormeventleu-AMP32.5–51.7(117) Afterstorminducedβ-d-glucosidase21.9–57.6 LaptevSeaArcticleu-AMP3.3(222) LenaRiverplumeArctic,eutrophicleu-AMP28.6–83.3(222) β-d-glucosidase14.3–40 Phosphatase8–28.6 NorthSeaCoastalzonewaterProteinase6.67(223) particularlyhighonorganicparticlesandindeepwater(Table2).Ingeneral,thecharacter- istics of these variables indicate (in some cases clearly) a dependency on the prevailing environmental conditions. III. FUNCTIONALITY OF EXTRACELLULAR ENZYMES SUBSTRATES IN MARINE ENVIRONMENTS A. The Size Continuum of Organic Matter from DOM to POM Dissolved organic matter (DOM) in the ocean is recognized as one of the three main reservoirs of organic matter on the planet, equal to the organic matter stored in terrestrial plants or soil humus (64). DOM of natural waters is chemically complex: less than 40% of the oceanic DOM pool is chemically characterized. The concentration of DOM is, therefore, frequently measured as dissolved organic carbon (DOC). DOC in the ocean typically decreases from the euphotic zone with concentrations ranging from 100 to 150 µM C to around 40 µM C in the ocean’s interior (65). Despite the lower concentrations in the deep ocean, the major fraction of the DOM is found in the aphotic zone of the ocean, comprising ϳ90% of the total oceanic DOC (66). Chemical characterization of oceanic DOM is hampered by both the low concentra- tions of DOM and the high salt content of ocean waters, which interferes with chemical analysis. Typically, 20–30% of oceanic DOC is recovered via 1000-Da ultrafiltration (67). In estuarine environments, recoveries of DOM are usually higher (up to 70%), as a result of the higher average molecular weight of the DOM in fresh water (68). On the basis of size fractionation studies performed over the past two decades on oceanic DOM, it appears that most of the DOM retained by ultrafiltration through 1000-Da filter cartridges is com- posed of compounds in the size range of 1000 to 30,000 Da (67–71). This high-molecular- weight DOM has been shown to be of contemporary origin (67,69) and derived from release processes taking place during photosynthesis of phytoplankton, grazing, and lysis of organisms (72–75). Phytoplankton extracellular materials have a similar carbohydrate Copyright © 2002 Marcel Dekker, Inc. signature to that of the oceanic DOM in surface layers (76–78). Phytoplankton activity and mortality (grazing and viral lysis) have been suggested to be the major source of oceanic DOM (70,74,75,78–82). The molecular weight fraction of the DOM larger than 1000 Da but smaller than 0.2 µm is also frequently termed colloidal organic matter (COC), in contrast to the truly dissolved DOM of Ͻ1000 Da. Freshly produced high-molecular-weight DOM consists mostly of carbohydrates, as indicated also by overall C:N ratios ranging from 15 to 25 (67,69,70). In addition to polysaccharides, proteins and lipids are present as chemically characterizable DOM components. Polysaccharides, however, are by far the most abundant macromolecular class of oceanic DOM. DOM is likely present as a size continuum in seawater; molecular weight and hydro- dynamic volume of DOM may vary with specific environmental conditions. For example, the fibrillar structure of polysaccharides allows them to form bundles of molecules bound together via cationic bridges mediated by Mg and Ca (83). Thus, coagulation processes of DOM, and particularly of the polysaccharides, are likely to be more important in oceanic seawater than in freshwater systems, as a result of the higher ionic concentrations in seawa- ter. These coagulation processes lead to the formation of colloidal and ultimately micropar- ticulate organic material; thus DOM may be transformed to POM. In this coagulation process, polysaccharides play a major role as a result of their relatively high concentration and the physicochemical characteristics of the fibrillar structure (84–86). In 1998 Chin, Orellana, and Verdugo showed that even low-molecular-weight DOM has the potential to coagulate spontaneously to form polymeric gels (87). These gels represent condensed organic matter at a higher concentration relative to that of the surrounding water and might therefore be of considerable importance for bacterioplankton (1) and enzymatic hydrolysis. Furthermore, these microgels might interact with other colloidal matter, forming distinct submicrometer particles that are ubiquitously present in seawater at concentrations of up to ϳ10 9 ml Ϫ1 (88–93). Whereas these submicrometer particles are not colonized by bacte- ria, the larger transparent exopolymer particles (TEPs) are frequently densely colonized by bacteria (94,95). TEPs have been shown to originate mainly from phytoplankton blooms and their decay (96). In addition to these polysaccharidic TEPs, protein particles have been reported to be abundant in the surface layers of the ocean (97). At the upper end of the size continuum of condensed colloidal organic matter, marine snow is commonly present, although at highly varying concentrations, in the surface as well as in the deep waters. The structural frame of this marine snow is also provided by polysaccharides: they are highly hydrated structures larger than 0.5 mm and range up to meters in diameter as observed in the subpycnocline layers of the Adriatic and Mediterra- nean Seas and in the deep ocean (24,98–100). Whether there are close links between different size categories of condensed organic matter and whether smaller aggregates are really the precursors for the next larger group of particles remain unclear at the moment. There are indications, however, based on the common chemical signatures of the polysaccharide pool (which dominates the macromo- lecular fraction of all these particles), that there is a link between them and that they are derived mainly from auto- and heterotrophic microorganisms. Irrespective of the exact relationships between submicrometer particles, TEP, and marine snow, all of this polysaccharide-based condensed matter interacts with the sur- rounding chemical environment by adsorbing inorganic and organic nutrients. This results in a higher nutrient concentration on these particles than in the ambient water (99,24). Copyright © 2002 Marcel Dekker, Inc. Thenutrient-enrichedzonesmightbeinthemicrometerrange,similartothemicrozones proposedbyAzam(1)andelegantlyvisualizedbyBlackburnetal.(101),ormarinesnow ofmetersindiameter.Inanycase,theyareattractivetoindigenousbacteriabecauseof nutrientconcentrationsuptothreeordersofmagnitudehigherthanintheambientwater (102,103).Similarly,bacteriaalsohavebeenreportedtobeenrichedbyuptothreeorders ofmagnitudeontheseparticles(24). B.EnzymeActivityandDOM/POMReactivity ContemporaryDOMofhighmolecularweighthasbeenshowntobeefficientlyutilizedby bacterioplankton(3,4),whilethemajorityofoceaniclow-molecular-weightDOM,which persistslongenoughtobemeasured,canbeconsideredasrefractory.Thisfindingledto theformulationofthesize-reactivitymodel(3,4)proposingthatthemajorityofthelow- molecular-weightDOMpoolistheconsequenceofchemicalandbiologicaldegradation (4).Sincebacterioplanktoncantakeupmoleculesonlysmallerthan600Dawithoutprior cleavagebyextracellularenzymes,theefficientutilizationofthishigh-molecular-weight DOMindicatestheimportanceofbacterialextracellularenzymes.Thisenzymepoolin- cludesbothendo-andexohydrolases(104).Endohydrolasescleavepolymersintooligo- mericcompounds,and,subsequently,exohydrolasesgeneratemonomers,whicharetaken upbybacteria.Inordertocleavecomplexmolecules,severalendohydrolasesactincon- cert,ashasbeendemonstratedforthecellulasecomplex(105).Withcommonlyused fluorogenicsubstrateanalogs,suchasmethylumbelliferylderivatives(13,21),onlythe finalstepofthecleavageofthemonomer(i.e.,theexohydrolaseactivity)canbemeasured. Themajorityofthiscleavageactivitybyhydrolasesisboundtothecellwalloroccurs intheperiplasmicspaceofGram-negativebacteria(106),andonlyasmallpercentageof freelydissolvedenzymaticactivitycanbedetected(seeSec.III.A.).Fluorescentlylabeled high-molecular-weightsubstrates(seeSectionIV.F.)canbeusedtomeasureendohydro- laseactivities. C.SignificanceofEnzymeActivityforSubstrateSupply Bacteriahavedifferentpossibilitiestorespondtonutrientlimitation(107)becausethey canuseinorganicaswellascombinedandmonomericorganicmoleculestosupplytheir cellulardemandsforenergy,growth,andmaintenance.Therelativecontributionofdiffer- entsourcestobacterialnutritiondependsessentiallyontheavailabilityofinorganicnutri- ents(108)andontheC:N:Pratios(e.g.,106:16:1;theRedfieldratio)oforganicmatter (109),whichdetermineitsnutritionalvalueafterhydrolysis.Examplesoftheutilization oftheNpoolsarepresentedinTable4.Using 15 NH ϩ 4 techniques, Tupas and Koike (110) demonstrated that natural bacterial assemblages in nutrient-enriched seawater cultures fu- eled 50–88% of their N demands for growth by NH ϩ 4 even in the presence of large amounts of DON. This DON consisted mostly of combined amino acids and contributed, together with NH ϩ 4 uptake, 70–260% of bacterial N production. However, an average of 80% of the DON used was subsequently remineralized to NH ϩ 4 by the bacteria (110). In general, information is too scarce to derive principles about the preferences of bacteria for specific N sources in the sea, however, hydrolysis of dissolved combined amino acids is always a prominent feature. Copyright © 2002 Marcel Dekker, Inc. [...]... Laminarin Xylan MUF-α-glucose MUF-β-glucose MUF-α-glucose MUF-β-glucose MUF-β-glucose MUF-α-glucose 115–175 115–175 115–175 37 34 27 37 34 27 1000 1000 439 –567 135 –1680 (161) b (161) b (161) b (1 43) (1 43) (146) (147) (202) ( 139 ) 0.1–0.7 MUF-β-glucose 135 –1680 0–0.79 0.08–2. 13 0–0.6 0 .3 0.9 0.0049–0.0082 0.002–0.016 ca 0.005–0.006 0.010–0.090 ca 0.50 ca 0 .30 MUF-α-glucose MUF-β-glucose MUF-α-glucose MUF-β-glucose... Activity in Coastal and Shallow Marine Sediments Rate (nmol cm 3 h Ϫ1) 90 114 216 240 660 139 2 1.6–8 .3 a 0.19–1.5 a 2.0 3. 5 a 0.41–1.2 a 0.58–0.94 a 0.41–0.86 a 4.7 2.6 0.90 0.41 0.64 0.22 Substrate Depth Site Reference MUF-β-fucose MUF-β-arabinose MUF-β-xylose MUF-β-mannose MUF-β-galactose MUF-β-glucose MUF-β-glucose Pullulan Laminarin Pullulan Laminarin Xylan Pullulan Laminarin Chondroitin sulfate Arabinogalactan... Comparing the enzymatic properties of three oceanographic provinces of the Pacific (northern subtropical, equatorial, and the Southern Ocean down to Antarctica), Christian and Karl (46) found significant variations in aminopeptidase and β-glucosidase activities and their temperature characteristics The relative relationship between aminopeptidase and β-glucosidase shifted from 0 .3 at the equator to 5 93 in. .. on enzymes from thermophilic and hyperthermophilic organism, and their potential use in biotechnological and industrial processes Probably the best-known examples of marine-derived enzymes in commercial applications are the DNA polymerases used in PCR, such as Pfu (derived from Pyrococcus furiosus; Stratagene) and Vent (New England Biolabs), derived from Thermococcus litoralis Hyperthermophilic enzymes. .. β-glucosidase in the water column of the Adriatic Sea, Rath and Herndl (187) detected only two different β-glucosidases However, using capillary electrophoresis, up to 8 different bacterial β-glucosidases were detected in a single sample and a total of 11 β-glucosidases during the wax and wane of the spring phytoplankton bloom in the coastal North Sea (188) Major changes in the diversity of the β-glucosidases... Cottrell and colleagues (190) investigated the chitinases of uncultured marine microbes by such a strategy They extracted DNA from seawater, then used a lambda phage cloning vector to produce libraries of genomic DNA These libraries were screened for chitin-hydrolyzing activity by using MUF-β-d-N, N′-diacetylchitobioside, and chitobiase activity was then assayed in protein extracts prepared from the positive... hydrolyzed to the same extent as proteins prepared by iodination of tyrosine residues, but very little of the methylated protein was ultimately assimilated or respired Taylor (1 73) measured the degradation of sorbed and dissolved proteins in seawater by the hydrolysis of [methyl3 H]–ribulose-1,5-biphosphate carboxylase-oxygenase (Rubisco) In another study, [32 P]– adenoshine triphosphate (ATP) (174) and the. .. 10:570–576, 1965 8 KM Khailov, ZZ Finenko Organic macromolecular compounds dissolved in sea-water and their inclusion into the food chains In: JH Steele, ed Marine Food Chains Edinburgh: Oliver & Boyd, 1970, pp 6–18 9 J Overbeck Early studies on ecto- and extracellular enzymes in aquatic environments In: ´ RJ Chrost, ed Microbial Enzymes in Aquatic Environments Berlin: Springer Verlag, 1991, pp 1–5 10 F... extracellular enzymes Microb Ecol 37 :86–94, 1999 126 DL Santavy, WL Jeffrey, RA Anyder, J Campbell, P Malouin, L Cole Microbial community dynamics in the mucus of healthy and stressed corals hosts Bull Mar Sci 54:1077–1087, 1994 127 SS Oosterhuis, MA Baars, WCM Klein-Breteler Release of the enzyme chitobiase by the Copyright © 2002 Marcel Dekker, Inc 128 129 130 131 132 133 134 135 136 137 138 139 140 141... mainly to an increase of β-glucosidase activity from 0.44 in Antarctica to 1519 nmol L Ϫ1 d Ϫ1 at the equator The authors hypothesized that there was a longitudinal trend in bacterial utilization of polysaccharides relative to amino acids and proteins Investigating the northern Pacific from 45°N 165°E down to the south edge of the equatorial zone (8°S 160°E), Koike and Nagata (119) also found an increase . Maine ( 137 ) 216 MUF-β-xylose Intertidal sediments Maine ( 137 ) 240 MUF-β-mannose Intertidal sediments Maine ( 137 ) 660 MUF-β-galactose Intertidal sediments Maine ( 137 ) 139 2 MUF-β-glucose Intertidal. (161) b 1.2 3. 8 a Laminarin 115–175 Arctic Ocean (161) b 0.04–0 .33 a Xylan 115–175 Arctic Ocean (161) b 0.02–0 .35 MUF-α-glucose 37 34 27 Arctic continental slope (1 43) 0.02 3. 02 MUF-β-glucose 37 34 27. (18,21 ,35 ,36 ) and probably also in the extracellular release of endo -enzymes by these bacteria (37 ,38 ). By hydrolyzing macromolecular linkages in an endo- fashion (i.e., hydrolyzing the nonterminal linkages in