Megazyme/AzoCarob Galactomannan/SACGLM/4 grams

Asimpleassayprocedureforβ-D-mannanaseenzymehasbeendevelopedwhichemployscarobD-galacto-D-mannandyedwithRemazolbrilliantBlue.Additionally,theprocedureisquantitative,relativelysensitive,andhighlyspecificforβ-D-mannanaseenzyme.Itcanbereadilyusedforthedeterminationofβ-D-mannanaseactivityincrudeenzymepreparationsandcolumn-chromatographyeluates.

ThegenomesequenceofthehyperthermophilicbacteriumThermotogamaritimaencodesanumberofglycosylhydrolases.Manyoftheseenzymeshavebeenshowninvitrotodegradespecificglycosidesthatpresumablyserveascarbonandenergysourcesfortheorganism.However,becauseofthebroadsubstratespecificityofmanyglycosylhydrolases,itisdifficulttodeterminethephysiologicalsubstratepreferencesforspecificenzymesfrombiochemicalinformation.Inthisstudy,T.maritimawasgrownonarangeofpolysaccharides,includingbarleyβ-glucan,carboxymethylcellulose,carobgalactomannan,konjacglucomannan,andpotatostarch.

Theprimaryobjectiveofthisstudywastoexaminetheeffectsofenzymetreatmentonchemicalcompositionsandproductionofreducingsugarsinpalm(Elaeisguineenis)kernelexpeller(PKE).PKEwasincubatedin80%watercontentat55°Cfor18hwithenzymeorwithoutenzyme(ascontrol).Crudeprotein(CP)andgrossenergy(GE)contentsofPKEwerenotsignificantlydifferent(P>0.05)betweenthecontrolandenzymetreatedPKE;averaged17.95%forCPand15.88MJ/kgforGE.However,etherextract(EE)neutraldetergentfiber(NDF),aciddetergentfiber(ADF),hemicelluloseandcellulosecontentsofenzymetreatedPKEdecreased(P<0.01) by="" approximately="" 34.6,="" 26,="" 20,="" 35.7="" and="" 22.1%,="" respectively,="" compared="" with="" the="" control.="" total="" content="" of="" reducing="" sugars="" (mannose,="" glucose="" and="" galactose)="" of="" the="" enzyme="" treated="" pke="" increased="" by="" approximately="" 200="" folds="" compared="" to="" the="" control=""><0.01). enzyme="" treated="" pke="" had="" higher=""><0.01) cellulase="" and="" mannanase="" activities="" but="" not="" α-galactosidase.="">

Solid-StateFermentation(SSF)ofAspergillusnigerFTCC5003onPalmKernelCake(PKC)isapracticalapproachtoupgradePKCintovalueaddedproduct.PresentstudywasconductedonAspergillusnigerFTCC5003growthprofileandmodelsthatareabletodescribethegrowthinSSFusingPKCsubstrate.DuetothedifficultiesofseparatingcellbiomassquantitativelyfromthesubstrateforSSFsystems,indirectmethodformeasurementofcellgrowthduringSSFofPKCbyAspergillusnigerFTCC5003wasstudiedbasedontheestimationofglucosamineandproteincontent.Preliminaryrelationshipsbetweenglucosamineandproteincontentstofungaldrycellweight(D_w)weredevelopedusingsimulatedhomogenousSSFdatausingglassbeadsassupportmaterials.BothglucosamineandproteincontentswerewellcorrelatedtothefungaldrycellweightinSSFonsupportmaterialsforproteinandglucosamine,respectively.TheequationsobtainedwereusedfortheestimationofcellbiomassprofileduringSSFofPKCfromthedataofglucosamineandproteinasgrowthindicatorstudy.Theestimatedfungaldrycellweightbasedonproteinconcentrationandβ-mannanaseactivityasmetabolicactivityformicrobialgrowthwerewellcorrelatedtoPKCdryweightwhich,indicatingthatbothweresuitableMarkerindescribingthegrowthofA.nigerFTCC5003inthissystem.Incontrast,estimatedfungaldrycellweightbasedonglucosamineconcentrationwasnotsuitabletodescribethegrowthofA.nigerFTCC5003.

Palmkernelcake(PKC)isusedwidelyintheanimalfeedindustrybuthaslimiteduseinpoultryfeedduetoitshighfibreandlowproteincontents.Themajorcomponentofthefibreismannanwhichisinsolubleanddifficulttodigest.Therefore,isolationoflocalpotentialmicrobescapableofdegradingmannanwasexploredfromvarioussourcessuchaspeatsoil,rottenoilpalmtrunksandrawPKC.TheisolateswerescreenedbasedontheclearingzonemethodonselectiveagarmediacontainingAzo-carob-galactomannanassubstrate.Atotalof36isolateswerescreenedandonly13hadaclearzonerangingfrom3.42±0.02mmto5.44±0.06mmwhichwereusedforfurtheranalysis.Theseisolateswereincubatedinshakeflasksat35°Cfor48hwith10g/litrePKCassubstrate.ThebestenzymesproducerwasisolateIBRLF16.A4withspecificmannanaseenzymeacitivityof17.82±0.05U/mg,productionofglucosamineat9.84±0.11mg/gandmannoseat9.54±0.06mg/g.TheisolateIBRLF16.A4wasidentifiedasAspergillusnigerIBRLF16.A4usingascanningelectronmicroscope.

CellulosomespreparedbythecelluloseaffinitydigestionmethodfromClostridiumthermocellumculturesupernatanthydrolysedcarobgalactomannanduringincubationat60°CandpH6.5.ArecombinantphageexpressingmannanaseactivitywasisolatedfromalibraryofC.thermocellumgenomicDNAconstructedinλZAPII.TheclonedfragmentofDNAcontainingaputativemannanasegene(manA)wassequenced,revealinganORFof1767nt,encodingaprotein(mannanaseA;Man26A)of589aawithamolecularmassof66816Da.Theputativecatalyticdomain(CD)ofMan26A,identifiedbygenesectioningandsequencecomparisons,displayedupto32%identitywithothermannanasesbelongingtofamily26.ImmediatelydownstreamoftheCDandseparatedfromitbyashortproline/threoninelinkerwasaduplicated24-residuedockerinmotif,whichisconservedinallC.thermocellumcellulosomalenzymesdescribedthusfarandmediatestheirattachmenttothecellulosome-integratingprotein(CipA).Man26AconsistingoftheCDalone(Man26A)washyperexpressedinEscherichiacoliBL21(DE3)andpurified.Thetruncatedenzymehydrolysedsolubleandinsolublemannan,displayingatemperatureoptimumof65°CandapHoptimumof6.5,butexhibitednoactivityagainstotherplantcellwallpolysaccharides.AntiserumraisedagainstMan26A’cross-reactedwithapolypeptidewithamolecularmassof70000DathatispartoftheC.thermocellumcellulosome.AsecondvariantofMan26AcontainingtheN-terminalsegmentof130residuesandtheCD(Man26A")boundtoivory-nutmannanandweaklytosolubleCarobgalactomannanandinsolublecellulose.Man26AconsistingoftheCDalonedidnotbindtothesepolysaccharides.TheseresultsindicatethattheN-terminal130residuesofmatureMan26Amayconstituteaweakmannan-bindingdomain.SequencecomparisonsrevealedalackofidentitybetweenthisregionofMan26Aandotherpolysaccharide-bindingdomains,butsignificantidentitywitharegionconservedinthethreefamily26mannanasesfromtheanaerobicfungusPiromycesequi.

Fungiareimportantforbiomassdegradationprocessesinmangroveforests.Giventhepresenceofseawaterintheseecosystems,mangrovefungiareadaptedtohighsalinity.HereweisolatePestalotiopsissp.NCi6,ahalotolerantandlignocellulolyticmangrovefungusoftheorderXylariales.Westudyitslignocellulolyticenzymesandanalysetheeffectsofsalinityonitssecretomes.Denovotranscriptomesequencingandassemblyindicatethatthisfunguspossessesofover400putativelignocellulolyticenzymes,includingalargefractioninvolvedinlignindegradation.Proteomicanalysesofthesecretomessuggestthatthepresenceofsaltmodifieslignocellulolyticenzymecomposition,withanincreaseinthesecretionofxylanasesandcellulasesandadecreaseintheproductionofoxidases.Asaresult,celluloseandhemicellulosehydrolysisisenhancedbutligninbreakdownisreduced.Thisstudyhighlightstheadaptationtosaltofmangrovefungiandtheirpotentialforbiotechnologicalapplications.

Theaimofthisstudyistoproduceandprofilethemannan-degradingenzymesfromlocalfungalisolatesbysubmergedfermentationandsaccharificationofPKC.ResultsshowedthatAspergillusnigerandSclerotiumrolfsiicanproducemannan-degradingenzymes.ThefungiweregrowninsubmergedfermentationofPKCtoproducemannan-degradingenzymes.Thehighestalpha-galactosidasewasobtainedonday13offermentation(0.128UmL^-1)whenusingA.nigerandonday18(0.126UmL^-1)whenusingS.rolfsii.Analysisalsoshowedthatenzymeactivitiesforbeta-mannanaseusingS.rolfsiiwerethehighestatday17(3.166UmL^-1)andforA.niger(2.482UmL^-1)atday8.Meanwhilethehighestbeta-mannosidasewereobtainedatday16forA.niger(0.128UmL^-1)andforS.rolfsiiatday16(0.116UmL^-1).

Crystalsofanendo-β-1,4-mannanase(1,4-β-D-mannohydrolase,E.C.3.2.1.78)fromPseudomonasfluorescenssubspeciescellulosahavebeengrownbythehanging-droptechniqueat291Koveraperiodofonetotwoweekstomaximaldimensionsof0.17×0.17×0.25mm.ThesecrystalsbelongtothespacegroupR32(orR3)withcelldimensionsofa=b=155.4andc=250.8A(hexagonalsetting)andcontainthree(six)moleculesintheasymmetricunit.Thecrystalsdiffracttoatleast3.2/kusingalaboratorysourceandaresuitableforstructuredetermination.

Thegenesman26aandman2AfromCellulomonasfimiencodemannanase26A(Man26A)andβ-mannosidase2A(Man2A),respectively.MatureMan26Aisasecreted,modularproteinof951aminoacids,comprisingacatalyticmoduleinfamily26ofglycosylhydrolases,anS-layerhomologymodule,andtwomodulesofunknownfunction.ExposureofMan26AproducedbyEscherichiacolitoC.fimiproteasegeneratesactivefragmentsoftheenzymethatcorrespondtopolypeptideswithmannanaseactivityproducedbyC.fimiduringgrowthonmannans,indicatingthatitmaybetheonlymannanaseproducedbytheorganism.AsignificantfractionoftheMan26AproducedbyC.fimiremainscellassociated.Man2Aisanintracellularenzymecomprisingacatalyticmoduleinasubfamilyoffamily2oftheglycosylhydrolasesthatatpresentcontainsonlymammalianβ-mannosidases.