Enzymes in the Environment: Activity, Ecology and Applications - Chapter 4 potx
Enzymes in the Environment: Activity, Ecology and Applications - Chapter 4 potx
... 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 ... the control. In contrast, the β-glucosidase, β-galactos-idase, and N-acetyl glucosaminidase activities decreased with the inoculation of the DAPGϩstrain(Table1).Theseresultsindicatethatsoilenzymesaresensitivetotheimpact ... 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...
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Enzymes in the Environment: Activity, Ecology and Applications - Chapter 8 potx
... 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 ... 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 ... 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...
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Enzymes in the Environment: Activity, Ecology and Applications - Chapter 9 potx
... free-living and marine-snow-associated bacteria. Mar Biol 113: 341 – 347 , 1992. 34. TK Kirk, RL Farrell. Enzymatic ‘‘combustion’’: The microbial degradation of lignin. AnnuRev Microbiol 41 :46 5–505, ... 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 ... bags. In using the ap-proach of Sinsabaugh et al. (73) and Jackson et al. (29), confined and in situ POM sampleswere assayed monthly for β-glucosidase, β-N-acetylglucosaminidase, β-xylosidase,phenol...
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Enzymes in the Environment: Activity, Ecology and Applications - Chapter 1 ppsx
... 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 ... by Sinsa-baugh and colleagues (200), who measured six enzyme ( -1 , 4- glucosidase, -1 , 4- endoglu-canase or endocellulase, -1 , 4- exoglucanase or exocellulase, β-xylosidase, phenol oxi-dase, and ... AGHutgasse 4, Postfach 812, CH -4 0 01 Basel, Switzerlandtel: 4 1-6 1-2 6 1-8 48 2; fax: 4 1-6 1-2 6 1-8 896World Wide Webhttp:/ /www.dekker.com The publisher offers discounts on this book when ordered in bulk...
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Enzymes in the Environment: Activity, Ecology and Applications - Chapter 2 pptx
... Inc.Currently,itisevidentthatmicroorganismsformcomplexmicrobialfoodwebsinallaquaticecosystems,andthattheiractivitiesandmetabolismsoftenaretightlycoupled and/ ormutuallyaffected(132, 143 , 144 ).Therefore,itisnotsurprisingthatenzymaticpropertiesandactivitiesofdifferentcomponentscreatingthemicrobialfoodwebsinlakeecosystemshavedemonstratedcloserelationships.Severalreportshavedocumentedthestrongdependencyofbacterialsecondaryproductiononectoenzymeactivitiesofaquaticmicroorganisms(2 4, 16,17,19,25,28,29,33,36,59).Thereoftenisasignificantcorrelationbetweenphytoplanktonprimaryproductionandactivitiesofdifferentectoenzymesinfreshwaterecosystems(25,28,29,33,52).Ourstudiesinlakesofdifferingdegreesofeutrophicationhaveshownmicrobialesteraseactivitytobepositivelycorrelatedtophytoplanktonprimaryproduction,bacterialsecondaryproduction,andconcentrationofdissolvedorganiccarbon(DOC)(Fig.13).Wehavefoundasignificantnegativerelationshipbetweenenzymeactivityandtheper-centageofphytoplanktonextracellularrelease(PER)ofphotosyntheticorganiccarboninthestudiedlakes.ThisnegativecorrelationbetweenPERandesteraseactivityindicatedthatenzymesynthesiswaspartiallyinhibitedinbacteriabylow-molecular-weightphoto-syntheticproductsofphytoplanktonthatwerereadilyutilizedbythesemicroheterotrophs:i.e.,catabolicrepressionofesterasesynthesiswasfoundinlakescharacterizedbyhighPERofphytoplankton(29,33).VIII.ECTOENZYMEACTIVITYANDLAKEWATEREUTROPHICATIONTheimportanceoforganicmatterasavariableforevaluatingthetrophicstatusoflakeshasbeenrecognizedsincethebeginningofthe20thcentury( 145 , 146 ).Increasingconcen-trationsoforganicconstituentsinwaterarethedistinctindicatorsofacceleratedeutrophi-cationprocessesinmanylakes( 147 – 149 ).OurstudiesclearlydemonstratedthatenzymeactivitiesweresignificantlypositivelyproportionaltoDOCcontentoflakes(Fig.13C).Asdescribedearlierinthischapter,severalmicrobialectoenzymesareresponsibleforrapidtransformationanddegradationofbothdissolvedorganicmatterandPOMinfresh-waterecosystems.Therefore,wehypothesizethatan‘‘enzymaticapproach’’canbeveryusefulinthestudiesoflakeeutrophication.Severalreportspointedoutthatmicrobialenzymaticactivitieswerecloselyrelatedtotheindicesofwatereutrophicationand/orthetrophicstatusofaquaticecosystems(25,27,29,31,33,38,52,58,62,78).Ourstudiesalongthetrophicgradientoflakes(fromoligo/mesotrophictohypereutrophiclakes[Fig.14A]supportourhypothesis(andtheassumptionsofothers)thatselectedenzymaticmicrobialactivitiesareverypracticalforarapidrecognitionofthecurrenttrophicstatusoflakes.Activitiesofalkalinephosphatase,esterase,andaminopeptidaseincreasedexponentiallyalongatrophicgradientandcorre-latedsignificantlywiththetrophicstateindexofthestudiedlakes(Fig.14B,C,D).Wealsofoundastrongrelationshipbetweenactivitiesofectoenzymesandphytoplanktonprimaryproductionintheselakes.RapidincreasesinectoenzymeactivitieswereobservedespeciallyinarangeofgraduallyeutrophiclakeswhenthevalueofCarlson’strophicstateindex(TSI)wasabove55(150)(Fig. 14) .Moreover, ... Inc.lakewater.Figures2Band2CshowthatectoenzymesynthesisinDOM-enrichedsampleswasnolongerrepressedwhentheconcentrationofthereadilyutilizablelowmolecular-weightmoleculesfellbelowacriticallevel,andpolymericsubstrateshadtobeusedtosupportthegrowthandmetabolismofbacteria.Similarinsituobservationsduringphyto-planktonbloomdevelopmentandbreakdownwerereportedforβ-glucosidaseactivityineutrophicLakePlußsee( 24) ,forβ-glucosidaseandaminopeptidaseactivitiesinmeso-trophicLakeScho¨hsee(25),andforlipaseactivityineutrophicLakeMikołajskie (40 ).Despitethewidespreadoccurrenceofcatabolicrepression,withtheexceptionofthoseforentericbacteria,themoleculardetailsoftherepressionarepoorlyunderstood.Somestudieshaveindicatedthatcyclicadenosinemonophosphate(cAMP),togetherwithitsreceptorprotein,mayplayacentralroleincontrolofcatabolicrepression (41 ,42 ).Usingtherepressionstrategyforectoenzymesynthesis,microorganismscanavoidthewastefulproductionofinducibleenzymes,whicharenotusefulwhentheirgrowthisnotlimitedbyUDOM(3,19, 24, 35).B.InhibitionofActivityItisimportanttoconsiderthattherepression/derepressionofanectoenzymenotbeequatedtothereversibleinhibitionofactivity.Evenifanectoenzymeissynthesized,itsactivitymaybeinhibitedbytheaccumulationoftheendproductorbyhighconcentrationsofthesubstrate(19).Twogeneraltypesofreversibleinhibitionareknown:competitiveandnoncompetitiveinhibition.Competitiveinhibitionoccurswhenaninhibitingcompoundisstructurallysimilartothenaturalsubstrateand,bymimicry,bindstotheenzyme.Indoingso,itcompeteswithanenzyme’snaturalsubstratefortheactivesubstrate-bindingsite.Thehallmarkofcompetitiveinhibitionofmanyectoenzymes(e.g.,alkalinephosphatase,β-glucosidase,aminopeptidase)isthatitdecreasestheaffinityofanectoenzyme(anincreaseoftheapparentMichaelisconstantisobserved)forthesubstrateand,therefore,inhibitstheinitialvelocityofthereaction(Fig.3)(13,26,37).Competitiveinhibitionisreversibleandcanbeovercomebyincreasedsubstrateconcentration,andthereforethemaximumvelocity(Vmax)ofthereactionisunchanged(Fig.3A).Noncompetitive ... the cyto-plasmic membrane, where they hydrolyze macromolecules in close vicinity to the cell. The resulting low-molecular-weight products are then transported across the cell mem-brane and utilized...
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Enzymes in the Environment: Activity, Ecology and Applications - Chapter 3 pdf
... α-glucosidase and aminopeptidase in marine environments. Mar Ecol ProgSer 1 14: 237– 244 , 19 94. 129. GA Vargo, E Shanley. Alkaline phosphatase activity in the red-tide dinoflagellate, Ptychodis-cus ... 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...
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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 ... high-molecular-weight fractions. Exohydrolases or -1 , 4- cellobiohydro-lases act only on the exposed ends of -1 , 4- glucan chains releasing the disaccharide cello-biose (17). β-Glucosidase and ... 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...
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Enzymes in the Environment: Activity, Ecology and Applications - Chapter 10 docx
... the years; these include vanilin, indulin, ferrulic acid, and, most importantly, 14 C-labeled synthetic lignins. Various fungal enzymes are involved in lignin degradation, including lignin peroxidase, ... wasconcluded that the higher levels of these 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 ... 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....
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Enzymes in the Environment: Activity, Ecology and Applications - Chapter 11 ppsx
... 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 ... by the clay of the substrate access to 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) ... (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...
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Enzymes in the Environment: Activity, Ecology and Applications - Chapter 12 ppsx
... systems, lasR-lasI and rhlR-rhlI. The lasI and rhlI gene productsare involved in the synthesis of two different AHL molecules, N-(3-oxododecanoyl)-l-homoserine lactone and N-buytryl-l-homoserine lactone, ... components in- clude β-galactosidase, β-N-acetylglucosaminidase, β-N-acetylgalactosaminidase, - and β-mannosidase, and α-fucosidase (116). Other bacteria then produce proteolytic enzymes, such ... hydrolytic enzymes into the soil, and these enzymes are capable of hydrolyz-ing pesticides. Degradation by fungal enzymes may be due to less specific enzymes, as in the case of lignin-degrading enzyme...
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