Purification and Characterization of a Collagenolytic Enzyme from a Pathogen of the Great Barrier Reef Sponge, Rhopaloeides odorabile 英文参考文献.docVIP

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Purification and Characterization of a Collagenolytic Enzyme from a Pathogen of the Great Barrier Reef Sponge, Rhopaloeides odorabile 英文参考文献.doc

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Purification and Characterization of a Collagenolytic Enzyme from a Pathogen of the Great Barrier Reef Sponge, Rhopaloeides odorabile 英文参考文献

PurificationandCharacterizationofaCollagenolytic EnzymefromaPathogenoftheGreatBarrierReef Sponge,Rhopaloeidesodorabile JoydeepMukherjee1,NicoleWebster2,LyndonE.Llewellyn2* 1SchoolofEnvironmentalStudies,JadavpurUniversity,Kolkata,India,2AustralianInstituteofMarineScience,Townsville,Queensland,Australia Abstract Background:Inrecentyearstherehasbeenaglobalincreaseinreportsofdiseaseaffectingmarinesponges.Whiledisease outbreaks have the potential to seriously impact on the survival of sponge populations, the ecology of the marine environmentandthehealthofassociatedinvertebrates,ourunderstandingofspongediseaseisextremelylimited. Methodology/PrincipalFindings:AcollagenolyticenzymesuspectedtoenhancepathogenicityofbacterialstrainNW4327 against the sponge Rhopaloeides odorabile was purified using combinations of size exclusion and anion exchange chromatography.Afterachievinga77-foldincreaseinspecificactivity,continuedpurificationdecreasedtheyieldto21-fold with7.2%recovery(specificactivity2575collagendegradingunitsmg21protein)possiblyduetoremovalofco-factors.SDS- PAGEofthepartiallypureenzymeshowedtwoproteinsweighingapproximately116and45kDawiththeheavierband being similar to reported molecular weights of collagenases from Clostridium and marine Vibrios. The enzyme degraded tissue fibres of several sponge genera suggesting that NW4327 could be deleterious to other sponge species. Activity towardscaseinandbirdfeatherkeratinindicatesthatthepartiallypurifiedcollagenaseiseitheranon-selectiveprotease abletodigestcollagenoriscontaminatedwithnon-specificproteases.EnzymeactivitywashighestatpH5(theinternalpH ofR. odorabile) and30uC(the average ambientseawater temperature). Activity underpartially anaerobic conditions also supports the role of this enzyme in the degradation of the spongin tissue. Cultivation of NW4327 in the presence of collagenincreasedproductionofcollagenaseby30%.EnhancedenzymeactivitywhenNW4327wascultivatedinmedia formulatedinsterilenaturalseawaterindicatesthepresen