Enzymes in the Environment: Activity, Ecology and Applications - Chapter 1 ppsx

... 4, Postfach 812 , CH-40 01 Basel, Switzerlandtel: 4 1- 6 1- 2 6 1- 8 482; 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 quantities. ... Environ 11 7 /11 8 :10 3 10 9, 19 92. 14 1. J-M Bollag, C Meyers, S Pal, PM Huang. The role of abiotic and biotic catalysts in the transformation of phenolic compounds. In: PM Huang, J Berthelin, J-M Bollag, ... 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 mineralizationhave...

... Inc.possibletofindseveralexplanationstointerpretaproteinadsorptionisotherm,withnoexperimentalevidenceavailabletochooseamongthem.TheadvantageoftheNMRmethodisthatitsimultaneouslygivesthequantityofadsorbedprotein,thesurfacecover-ageofthesolidbytheprotein,andthemonolayerormultilayermodeofadsorption (16 ).Onlyknowledgeofthesethreefactorsallowsapossibleunfoldingoftheproteinsontheclaysurfacestobedetectedandquantified. 1. NuclearMagneticResonanceDetectionoftheExchangeofaParamagneticCationonProteinAdsorptiononClaysTheprincipleofthemethod (16 )isbasedonthefactthattheadsorptionofproteinsonclayscausesthereleaseofcharge-compensatingcations(7 ,17 ).ItalsousesthesensitivityoftherelaxationtimesT 1 andT2ofnuclearspinstoparamagneticcationsinNMRspectros-copy (18 ,19 ).Asmallquantity(between 3and2 0µMdependingonthepH)ofaparamagneticcation,Mn2ϩ,isaddedtoasodium-saturatedmontmorillonitesuspension(1gL 1 )witha10-mMconcentrationoforthophosphate.Thesuspensionisstudiedby 31 PNMRspec-troscopy.Aninterestingphenomenonisobserved: (1) theMn2ϩcationsthatareadsorbedontheclaysurfacedonotinteractatallwiththeorthophosphate,asshownbythecompari-sonbetweentheclaysuspensionandsupernatantafterremovaloftheclaybycentrifuga-tion ;and( 2)theMn2ϩcationsinsolutioninteractwiththeorthophosphate,leadingtoalinearincreaseofthelinewidthathalfheight,∆ν 1/ 2,oftheorthophosphatepeakontheNMRspectrum.Thislasteffectistheresultoftheparamagneticcontributiontothede-creaseofthespin–spinrelaxationtime,T2,oftheorthophosphatesignal.Whenagivenquantityofproteinisintroducedintothissuspension,itdisturbstheequilibriumbetweentheparamagneticMn2ϩadsorbedontheclaysurfaceandthatinsolution.Theanalysisoftheresultinglinewidthoftheorthophosphosphatesignalgivesthequantityofcationsexchangedonadsorption.Witha300-MHzNMRspectrometer,themeasurementtakesafewminutes;witha500-MHzspectrometer,1minissufficient(evenlessifhigherconcentrationsofortho-phosphateareused).Asnocentrifugationisrequiredwiththismethod,thisshorttimeofsignalacquisitioniscompatiblewithkineticstudies.Theresultsareexpressedas∆νP,whichisthedifferencebetween∆ν 1/ 2inthesystemwithparamagneticcationsand∆ν 1/ 2inacontrolofthesamecomposition,(butwithoutparamagneticcations)dividedbytheconcentrationofparamagneticcations.ThesurfacecoverageoftheclaybytheproteincanbededucedfromthefractionofMn2ϩreleased.Theknowledgeofboththequantityofproteinadsorbedandthesurfacecoverageofthesolidallowsthecalculationoftheinterfacialareaofcontactbetweenasingleproteinmoleculeandtheclaysurfaceatdiffer-entpHandionicstrengths.2.ConformationalChangesonAdsorptionofaSoftProtein,BovineSerumAlbumina.DescriptionoftheProgressiveSurfaceCoverageoftheClayFigure1shows the ... Inc.possibletofindseveralexplanationstointerpretaproteinadsorptionisotherm,withnoexperimentalevidenceavailabletochooseamongthem.TheadvantageoftheNMRmethodisthatitsimultaneouslygivesthequantityofadsorbedprotein,thesurfacecover-ageofthesolidbytheprotein,andthemonolayerormultilayermodeofadsorption (16 ).Onlyknowledgeofthesethreefactorsallowsapossibleunfoldingoftheproteinsontheclaysurfacestobedetectedandquantified. 1. NuclearMagneticResonanceDetectionoftheExchangeofaParamagneticCationonProteinAdsorptiononClaysTheprincipleofthemethod (16 )isbasedonthefactthattheadsorptionofproteinsonclayscausesthereleaseofcharge-compensatingcations(7 ,17 ).ItalsousesthesensitivityoftherelaxationtimesT 1 andT2ofnuclearspinstoparamagneticcationsinNMRspectros-copy (18 ,19 ).Asmallquantity(between 3and2 0µMdependingonthepH)ofaparamagneticcation,Mn2ϩ,isaddedtoasodium-saturatedmontmorillonitesuspension(1gL 1 )witha10-mMconcentrationoforthophosphate.Thesuspensionisstudiedby 31 PNMRspec-troscopy.Aninterestingphenomenonisobserved: (1) theMn2ϩcationsthatareadsorbedontheclaysurfacedonotinteractatallwiththeorthophosphate,asshownbythecompari-sonbetweentheclaysuspensionandsupernatantafterremovaloftheclaybycentrifuga-tion ;and( 2)theMn2ϩcationsinsolutioninteractwiththeorthophosphate,leadingtoalinearincreaseofthelinewidthathalfheight,∆ν 1/ 2,oftheorthophosphatepeakontheNMRspectrum.Thislasteffectistheresultoftheparamagneticcontributiontothede-creaseofthespin–spinrelaxationtime,T2,oftheorthophosphatesignal.Whenagivenquantityofproteinisintroducedintothissuspension,itdisturbstheequilibriumbetweentheparamagneticMn2ϩadsorbedontheclaysurfaceandthatinsolution.Theanalysisoftheresultinglinewidthoftheorthophosphosphatesignalgivesthequantityofcationsexchangedonadsorption.Witha300-MHzNMRspectrometer,themeasurementtakesafewminutes;witha500-MHzspectrometer,1minissufficient(evenlessifhigherconcentrationsofortho-phosphateareused).Asnocentrifugationisrequiredwiththismethod,thisshorttimeofsignalacquisitioniscompatiblewithkineticstudies.Theresultsareexpressedas∆νP,whichisthedifferencebetween∆ν 1/ 2inthesystemwithparamagneticcationsand∆ν 1/ 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...

... 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 (11 6). Other bacteria then produce proteolytic enzymes, such ... Thiobacilli within pH-neutral and acidic minetailings and their role in the development of secondary mineral soil. Appl Environ Microbiol58 :19 04 19 12, 19 92. 11 0. G Southam, TJ Beveridge. Examination...

... Inc.Currently,itisevidentthatmicroorganismsformcomplexmicrobialfoodwebsinallaquaticecosystems,andthattheiractivitiesandmetabolismsoftenaretightlycoupled and/ ormutuallyaffected (13 2 ,14 3 ,14 4).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 (14 5 ,14 6).Increasingconcen-trationsoforganicconstituentsinwaterarethedistinctindicatorsofacceleratedeutrophi-cationprocessesinmanylakes (14 7 14 9).OurstudiesclearlydemonstratedthatenzymeactivitiesweresignificantlypositivelyproportionaltoDOCcontentoflakes(Fig .13 C).Asdescribedearlierinthischapter,severalmicrobialectoenzymesareresponsibleforrapidtransformationanddegradationofbothdissolvedorganicmatterandPOMinfresh-waterecosystems.Therefore,wehypothesizethatan‘‘enzymaticapproach’’canbeveryusefulinthestudiesoflakeeutrophication.Severalreportspointedoutthatmicrobialenzymaticactivitieswerecloselyrelatedtotheindicesofwatereutrophicationand/orthetrophicstatusofaquaticecosystems(25,27,29, 31, 33,38,52,58,62,78).Ourstudiesalongthetrophicgradientoflakes(fromoligo/mesotrophictohypereutrophiclakes[Fig .14 A]supportourhypothesis(andtheassumptionsofothers)thatselectedenzymaticmicrobialactivitiesareverypracticalforarapidrecognitionofthecurrenttrophicstatusoflakes.Activitiesofalkalinephosphatase,esterase,andaminopeptidaseincreasedexponentiallyalongatrophicgradientandcorre-latedsignificantlywiththetrophicstateindexofthestudiedlakes(Fig .14 B,C,D).Wealsofoundastrongrelationshipbetweenactivitiesofectoenzymesandphytoplanktonprimaryproductionintheselakes.RapidincreasesinectoenzymeactivitieswereobservedespeciallyinarangeofgraduallyeutrophiclakeswhenthevalueofCarlson’strophicstateindex(TSI)wasabove55 (15 0)(Fig .14 ).Moreover, ... EHP inhibitionaLake (m) Pi/EHP (%)Constance2 1. 39 1. 64 0.85 77 10 1. 80 1. 67 1. 08 80 12 1. 79 1. 05 1. 70 8220 6.70 2.08 3.22 91 50 28.49 0.80 35. 61 97 19 0 59.30 4.53 13 .09 94Schleinsee 1 1.30 ... sn-glycerol-3-phosphate and outer membrane protein Ic (e, E) with alkaline phosphatase and phosphate binding protein. J Bacteriol 14 3 :14 2 15 0, 19 80. 91. RT Heath, AC Edinger. Uptake of 32P-phosphoryl...

... 218 –478 6.9–25.0 (2 21) HT 18 8–625 5.4 12 .9San Francisco Bay Cells 1 µm 7.2 12 0 .16 –0.57 (18 )All cells 16 – 31 0.47 1. 60Uranouchi Inlet, Surface water 23.2 10 17 (11 4)Japan Bottom water 21. 1–270LT, ... essayUptake(nM h 1 ) Uptake(nM h 1 ) Uptake(nM h 1 )Delaware Estuary Salinity gradient 11 4 11 7 n.d. 14 C-AA, algal protein (17 1)LMW-DOC HMW-DOC% uptake d 1 % uptake d 1 Gulf of Mexico, ... anomeric linkages (16 0 16 2). A further technical advantage is that the fluorophoresused for labeling polysaccharides (fluorescein-amine, isomer II) and peptides (4-amino-3,6-disulfo -1 , 8-naphthalic...

... 0.6 71 17 549 53 388 27 59 1. 2 7.2 7.2Cont 2.2 61 12 480 30 208 19 27 1. 2 6.9 4.7Cont 1. 624 17 396 41 210 19 24 0.6 6.9 5.4F 113 1. 2 41 15 665 44 211 27 15 1. 6 6.9 6 .1 F 113 2.526 14 593 31 197 25 ... Inc.Althoughthisstudyinvolvedtheuseofageneticallymodifiedmicrobe,themodi - cationswerenotintendedtohaveafunctionalimpact;theywereinsertedasgeneticmark-ers.Asecondstudycomparingtheeffectofthesamegeneticallymarkedstraintothatofafunctionallymodifiedstrainshowedeffectsthataremoreinteresting(36).Theaimofthisworkwastodeterminetheimpactintherhizosphereofwildtypealongwithfunction-allyandnonfunctionallymodifiedPseudomonasfluorescensstrains.Thewild-typeF 113 straincarriedageneencodingtheproductionoftheantibiotic2,4-diacetylphloroglucinol(DAPG),usefulinplantdiseasecontrol,andwasmarkedwithalacZYgenecassette .The firstmodifiedstrainwasafunctionalmodificationofstrainF 113 withrepressedproductionofDAPG,creatingtheDAPGnegativestrainF 113 G22.Thesecondpairedcomparisonwasanonfunctionalmodificationofwild-type(unmarked)strainSBW25,constructedtocarrymarkergenesonly,creatingstrainSBW25EeZY-6KX.Significantperturbationswererecordedintheindigenousbacterialpopulationstruc-ture;theF 113 (DAPGϩ)straincausedashifttowardslower-growingcolonies(Kstrate-gists)comparedwiththenon-antibiotic-producingderivative(F 113 G22)andSBW25strains.TheDAPGϩstrainalsosignificantlyreduced,incomparisonwiththoseoftheotherinocula,thetotalPseudomonassp.populations,butdidnotaffectthetotalmicrobialpopulations.ThesurvivalofF 113 andF 113 G22wasanorderofmagnitudelowerthanthatoftheSBW25strains.TheDAPGϩstraincausedasignificantdecreaseintheshoot-to-rootratioincomparisontothatofthecontrolandotherinoculants,indicatingplantstress.F 113 increasedsoilalkalinephosphatase,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 F 113 (DAPGϩ) strain was...

... Inc.ThepatternsofenzymeactivityandmRNAaccumulationsuggestthatchitinases and -1 ,3-glucanasesmightbepartoftheearlydefenseresponsebytheplanttotheinvad-ingfungus,whichisthensuppressedassymbioticinteractionsdevelop.Inthiscontext,planthydrolasesmaybeinvolvedintheregulationofAMdevelopment.Nevertheless,someexperimentaldatarevealedthatitisnotlikelythatplantchitinasesandglucanasesareessentialtothecontrolofthegrowthofAMfungi.TransgenicplantsconstitutivelyexpressinghighlevelsofdifferentacidicformsoftobaccoPRs(includingchitinasesand -1 ,3-glucanases)becamenormallycolonizedbytheAMfungi (12 2 ,12 3).Thefactthatchitinasesand -1 ,3-glucanasesinducedbytheAMsymbioticfungiorbyconstitutivegeneexpression,donotpreventrootcolonizationsuggeststhattheyareineffectiveincontrollingfungaldevelopment.ThelowenzymaticaffinityforAMfungalcomponentsorinaccessibilityoftheseenzymestofungalcellwallcomponentsmaycausethisineffec-tiveness (11 2).Conversely,specificacidicformsofchitinaseand -1 ,3-glucanaseareactivatedinseveralplantscolonizedbyAMfungi.Thesesymbiotic,specificisoenzymeshavebeenreportedinpea (12 4),tobacco (11 8),andtomato (12 5 12 7)rootsandaredifferentfrompathogen-inducedisoformsorconstitutiveenzymes.Inaddition,newchitosanaseisoformshavebeenshowninpea (12 8)andtomato (12 6).Chitosanasesarehydrolyticenzymesactingonchitosan,aderivativepartiallyorfullydeacetylatedofchitin (12 9).Interestingly,themycorrhizal-relatedchitinaseisoformdescribedintomato-colonizedrootsappearedtodisplaychitosanaseactivity.Thisbifunctionalcharacterwasnotfoundfortheconstitutive enzymes, orinPhytophthorasp.–inducedchitinases (12 6).Mycorrhizal-specificplantchi-tinasesarenotactiveinpathogen-infectedroots (11 8 ,12 4 12 5)orinRhizobiumsp.legumesymbiosis (13 0),indicatingadifferentialinductionandfunction.AlthoughtheprecisefunctionofplanthydrolaseactivitiesintheestablishmentofAMsymbioticinteractionisstillunclear,theirstimulationseemstobeakeypointinthemechanismofrecognitionandsignalingbetweenplantrootsandAMfungi.AregulatoryroleoftheseenzymesduringestablishmentofAMandotherrootsymbiosishasbeenproposed.Stimulationofspecificplantchitinaseshasbeenreportedinsoybean/Rhizobiumsp. (13 1)andectomycorrhiza (13 2).Ithasbeenpostulatedthatchitinasesmaybeinvolvedintherecognitionoftherhizobialnodulationsignalsand,thus,intheregulationofthenodulationprocess (13 3).Thedatasuggestaspecificrolefortheseenzymes,onethatcouldberelatedintheAMsymbiosistothedetection,modification ,and/ orreleaseofchitinorchitosanoligomersfromthefungalcellwallthatcanactassignalingcompoundsduringthedevelopmentofAM(Fig.3).Inthisprocessofsignalexchange,themodulationof ... Inc.directlycontributetoreductionofpathogenviabilityandgrowth.Inaddition,theyhavebeenproposedasmediatorsinpathwaysleadingtodefense-relatedgeneexpression (13 6).ThereleaseofAOSinsomeplant–pathogeninteractionscanresultindamagetothehosttissues.Therefore,mechanismsthatlimitthedurationoftheoxidativeburstanditstoxiceffectsarenecessarytominimizedamagetotheplantitself.Oneofthesemecha-nismsistheactionofendogenousantioxidantenzymes,suchassuperoxidedismutases,catalases,peroxidases,andglutathioneperoxidases,whicharecapableofneutralizingtheAOS.Duringtheestablishmentofacompatibleplant–fungusAMsymbiosis,thehostplantshowedlittlereactionatthecytologicalleveltoappressoriumformationorinfectionhyphae.Occasionallysomethickeningwasobservedinepidermalcellwallsatthepointofcontactwithappressoria (10 5),andonlyaresponsesimilartoHRhasbeendetectedinRiT-DNA–transformedrootsofalfalfacolonizedbyGigasporamargarita (13 7).Nev-ertheless,recentstudies,usingthediaminobenzidine(DAB)stainingtechnique,revealedthatabrownishstain,indicativeofH2O2accumulation,waspresentwithincorticalrootcellsinthespaceoccupiedbyclumpedarbusculesandaroundhyphaltipsattemptingtopenetraterootsofMedicagotruncatulacolonizedbyG.intraradices (13 8).TheseresultssuggestthatalocallyrestrictedoxidativeburstcouldbeinvolvedintheresponseoftheplanttoAMformationanddevelopment.Relativelyfewdataexistconcerningthepossibleparticipationofantioxidanten-zymesintheplantresponsetoAMformation.Apeakofcellwall–boundperoxidasewasobservedduringtheinitialstagesoffungalpenetrationinleek(Alliumporrum)cells.Onceinfectionwasestablished,theactivitydecreasedtothelevelsshowninnonmycorrhizalplants (13 9).Inpotatoroots,theactivityofextracellularperoxidaserecoveredinrootleachateswasnotstimulatedbyAMinfection;peroxidaseactivitypergramoffreshweightwassignificantlyenhancedinAMroots (14 0).WhenpotatoplantsweregrownwithhigherPsupply,extracellularperoxidaseactivityincreasedlinearlywithincreasingPsupply,suggestingaroleofperoxidaseinlimitingAMinfectioninwell-P-nourishedplants (14 0).Theanalysisofcatalaseandascorbateperoxidaseactivitiesduringtheearlystageoftobacco–Glomusmosseaeinteractionrevealedtransientenhancementsofbothenzymaticactivitiesintheinoculatedplants (14 1).Theseincreasescoincidedwiththestageofappre-ssoriaformationonrootsurfacesandtheappearanceofapeakofaccumulationoffreesalicylicacidininoculatedroots (14 1).Thesedataindicatetheactivationofcatalaseandperoxidaseactivitiesinrootcellswherethefungusformingappressoriamightbepartoftheplantresponsetotheinvadingfungus.Theroleoftheseenzymesinthisresponsecouldberelatedtoactivationofadefensivemechanismortoaprocessofcellwallrepairatthesiteofinfection(Fig.3).Alternatively,theearlyactivationofcatalaseandperoxidasemay ... 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.(nitrificationanddenitrificationeffects)aswellasbyprotectionandcreationofwetlands(4,7,39,57,85 ,12 2).Itisagainstthisbackdropofthemajorenvironmentalrelevanceoftheenzymesofnitrogenandcarboncyclingprocessesthatthischapterispresented.Theutilityofsoilenzymeactivitiesasindicatorsofsoilqualityandinmonitoringoftheeffectsofsoilpollutionispresentedelsewhere (14 ,34,60 ,11 6 ,13 1)andinChapters15 ,16 ,and1 7 .The general objective of this chapter is to highlight the current status ... Soil Biol Biochem 10 :18 7 19 1, 19 78.8. J-M Bollag. Decontaminating soil with enzymes: An in situ method using phenolic and ani-linic compounds. Environ Sci Technol 26 :18 76 18 81, 19 92.9. H Bolton ... Enzymatic changes in the rhizosphere of loblolly pineexposed to ozone and acid rain. Soil Biol Biochem 23 :11 15 11 19, 19 91. 10 2. GB Reddy, RA Reinert, G Eason. Loblolly pine needle nutrient and soil enzyme...

... Laczko, W Matthey. Organic matter cycling in grassland soils of the Swiss Jura mountains: Biodiversity and strategies of the living communities. Soil Biol Bio-chem 31: 12 81 12 93, 19 99.47. WL ... Springer-Verlag, 19 91, pp 25–59. 10 . JS Clein, JP Schimel. Reduction in microbial activity in birch litter due to drying and rewettingevents. Soil Biol Biochem 26:403–406, 19 94. 11 . JS Clein, ... activity and secondary production in free-living and marine-snow-associated bacteria. Mar Biol 11 3:3 41 347, 19 92.34. TK Kirk, RL Farrell. Enzymatic ‘‘combustion’’: The microbial degradation of lignin....

... 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, ... Pflanzenerna¨hr Bodenk 15 4: 17 1 17 5, 19 91. 30. HTS Boschker, TE Cappenberg. A sensitive method for using 4- methylumbelliferyl-β-cellobi-ose as a substrate to measure (1, 4 )- -glucanase activity in sediments. ... 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....