Phialophora melinii NFCCI 3617 A Newly Isolated Psychrotolerant Fungus That Produces Enhanced LaccWord文档下载推荐.docx

Phialophora melinii NFCCI 3617 A Newly Isolated Psychrotolerant Fungus That Produces Enhanced LaccWord文档下载推荐.docx

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　　Apsychrotolerantfungus，isolatedfromdecomposingpineneedledebris，isinvestigatedforlaccaseproductionundertheinfluenceof5organicsolvents.ThefunguswasidentifiedasPhialophorameliniiandwasabletogrowbetween4to35°

C（opt.25°

C）and2-14pH（opt.5-7）.InquantitativeestimationsthatwerecarriedoutatoptimumgrowthtemperatureandpH，thefungallaccasewasestimatedtobe21.0±

4.0U/L.NativePAGEstudyrevealed35kDamolecularmassofthefungallaccase.Supplementationoforganicsolventsnamely，methanol，ethanol，acetone，n-propanolandiso-propanolinvaryingconcentrations（0.5%-2.0%，separately），significantlyaffectedtheproductionoffungallaccase.Outof5solventsused，n-propanolwasfoundtobethemostefficientenhanceroflaccaseproduction.n-Propanol（0.5%）resultedinmaximumenhancement（7folds）inlaccaseproductionat18thdayofincubation.Methanol，iso-propanolandethanolwereabletoenhancelaccaseproductionupto5-6foldsincomparisontocontrolwithrespecttothevaryingconcentrationandincubationlength.Ageofthefungalculture（incubationdays）wasobservedasanimportantfactorforlaccaseproduction.Useoflowmolecularcompoundsinenhancingthefungallaccaseproductionmaybeconsideredasaneco-friendlyapproach.

　　Keywords：

Phialophora；

biodegradation；

Laccase；

OrganicSolvents；

IndianHimalayanRegion

　　INTRODUCTION

　　Laccasesarethememberofoxido-reductasegroupofenzymeswithawidevarietyofapplications.Theycanoxidizeseveralaromaticcompoundsbyusingoneelectronmechanismthatfurtherleadstotheformationofwaterfrommolecularoxygen.Laccasesarewidelystudiedinfungiandplants，althoughtheyarealsoreportedfrombacteriaandinsects.Fungifrombasidiomycota，ascomycotaanddeuteromycotaphylaarethesourceoffungallaccases，however，basidiomycotaarethebeststudiedones.Ascomycotaanddeuteromycotaarealsoemergingassourceoflaccasesfromvariousecologicalniches.Laccasesareknownfortheirroleinthephysiologyofthefungi，theirapplicationsinindustriessuchastextile，food，pharmaceuticalsandsyntheticchemistry，andasaneco-friendlyapproachwithrespecttotheconceptof“greenchemistry”'

（Rodgersetal.，2010；

Jeonetal.，2012；

Rivera-Hoyosetal.，2013）.　　Laccasesfromthefungalsourceshavebeenwidelystudiedwithrespecttobiodegradation，lignindegradationinparticular（Novotnyetal.，2004；

Strong&

Claus，2011）.Temperatureisoneofthemajorgoverningfactorsinalltheecologicalprocesses.Inlowtemperatureenvironments，suchasmountainecosysteminIndianHimalayanRegion（IHR），degradationisaslowprocess.Dependingontheforesttypes，suchaspineforest，degradationisfurtherslowduetotherecalcitrantnatureofligninpolymerfoundinpineneedlesthatpersistsforlongtermandaffectsthebiologicaldiversityofsoil（Rizvi&

Rizvi，1992）.Thepsychrotolerantmicrobialcommunities，inhabitingtheseenvironmentstaketheresponsibilityofvariousbiologicalprocesses，includingbiodegradation（Mishraetal.，2012；

Singhetal.，2012；

Dhakar&

Pandey，2013；

Yarzá

bal，2014）.Thepsychrotolerantfungiproducecoldtolerantenzymesthatmayberelativelyinsmalleramountsbut，atthesametime，withtheactivityforlongerperiods（Dhakaretal.，2014a）.

　　Inviewoftheapplicationsoflaccasesavarietyofsupplementshavebeenusedasenhancersoflaccaseproduction（Kunamnenietal.，2007；

Bertrandetal.，2012）.Antibiotics，vitamins，aminoacidsandseveralaromaticcompoundshavebeenreportedtoaffectthelaccaseproduction（DhawanandKuhad，2002；

Dhakaretal.，2014a）.Amongaromaticcompounds，guaiacol，2，5-xylidineand2，6-dimethoxyphenolhavebeenreportedasinducerswhilecompoundssuchasTNT（2，4，6-trinitrotoluene）and1，4-hydroquinonehavebeenfoundtoactvariably（enhancerorinhibitor）onlaccaseproductionfromCerenaunicolor，GanodermalucidumandTrametesversicolor（Bettinetal.，2014）.Thisindicatestowardstheimportanceofscreeningofsupplementsfortheirroleinlaccaseproductionfromspecificsources.TheaimofthepresentstudyisthecharacterizationofacoldandpHtolerantstrainofPhialophorameliniiisolatedfromdegradingpineneedleswithrespecttoitslaccaseproductionpotentialunderinfluenceoflowmolecularweightcompounds.

　　1.MATERIALSANDMETHODS

　　2.1SiteDescription

　　Soilsampleswithdegradingneedleswerecollectedfromthepineforest（29°

37'

56"

N79°

20'

16"

E，2480mamsl，Soni-Binsar，Dist.Almora，Uttarakhand，India）.Thesiteexperiencesheavyrainfallduringmonsoonandoccasionalsnowfallduringwinterseason.ThesoilpHwas5.1±

0.5.

　　1.2TheFungus

　　Thefunguswasisolatedfollowingthestandardmethod（serialdilution）.MorphologicalandphysiologicalcharacterizationwasdoneonPotatoDextrose（PD）mediumat25?

C.Microscopicobservationswererecordedat100X（eyepiece）usinglacto-phenolcottonbluestain.SpecieslevelidentificationofthefunguswasprovidedbyNationalFungalCultureCollectionofIndia，AgharkarResearchInstitute，Pune.ThefungushasbeenassignedtheAccessionno.NFCCI3617.ThetemperatureandpHtolerancewasperformedonPDbetween4to35?

Cand1.0to14.0，respectively.　　1.3LaccaseProduction

　　ThefungalculturewasgrowninmodifiedKirk&

Farrell（1987）mediumsupplementedwithABTS（2，2'

-azino-bis（3-ethylbenzothiazoline-6-sulphonicacid）asdescribedinDhakarandPandey（2013）.TheABTSplateassayswereperformedat4，9，14，25and35?

C.Ligninolyticefficiencywascalculatedbyzonediameter（mm）/colonydiameter（mm）*100.QuantitativeestimationoflaccaseactivitywasdonebyABTSassayat420nm（Hanetal.，2005）usingspectrophotometricmethod.Reactionmixturecontainedcrudeenzyme，citratephosphatebuffer（pH4.5）andABTS（2mM），itwasincubatedatroomtemperaturefor2minandabsorbancewasrecordedat420nm.Enzymeactivitywasdefinedas1?

MofABTSoxidizedpermin.

　　1.4Molecularmassoflaccase

　　MolecularmassoflaccasewasdeterminedbyperformingnativePAGE.Chilledacetonewasaddedtocrudeenzymeandkeptat-20?

Cforovernight.Thenitwascentrifugedat8000rpmfor20minat4?

C.Pelletswerere-dissolvedinthebuffer（pH4.0±

0.5）andsupernatantwasdiscarded.Thepolyacrylamidegelconstitutedofseparating（12.5%）andstackinggel（4.0%）accordingtoLaemmli（1970）withoutSDS.Thegelwasincubatedatroomtemperaturefor30mininthecitrate-phosphatebufferofpH4.0±

0.5followingelectrophoresis.ItwasthentransferredtotheABTSsolution（0.5%）inthesamebufferandincubatedatroomtemperaturefor1h.GreenbandsappearedbyoxidationofATBSthroughlaccase.

　　1.5InfluenceOfLowMolecularWeightOrganicSolventsOnLaccaseProduction

　　Productionoflaccasewasinvestigatedundertheinfluenceoflowmolecularweightorganicsolvents.Laccaseproductionmediumwaspreparedbytaking50mlin250mlErlenmeyerflaskswithpH5.5±

0.5.5mmdiscs（1perflask）ofsixdaysoldculturewereusedforinoculationthatwasgrownonPDAat25?

C.Inoculatedflaskswereincubatedat25?

C.Fiveorganicsolvents（methanol，ethanol，acetone，n-propanolandiso-propanol）weresupplementedbetween0.5to2.0%at4thdayofinoculation，separately.Theseflaskswereincubatedat25?

Cinstaticconditions.Laccaseactivitywasdeterminedatevery6thdayofinoculationupto30thday.CulturebrothwasfilteredwithWhatmanno.1filterpaperandusedascrudeenzymeforfurtherestimations（ABTSassays）.

　　1.6StatisticalAnalysis

　　Alltheexperimentsweredoneintriplicates.OnewayANOVAwithposthocTukey’stestwasusedtoanalysetheresults.Thesignificancelevelwas5%（p　　2.RESULTS

　　2.1Morphology，GrowthCharactersAndLigninolyticEfficiencyOfTheFungus

　　ThefungusproducedwhitespikycolonyonPDagarat25?

Cfollowing5daysofincubation.Themicroscopicexaminationrevealedthedevelopmentofseptatemyceliumwithsingle，ovalshapedspores（2-3?

m）.Thefunguswasabletogrowbetween4to35?

C（optimumgrowthtemperature25?

C）and2to14pH（optimum5-7）.Basedoncolonymorphologyandmicroscopicfeatures，thefunguswasidentifiedasPhialophoramelinii.ThefungusproducedgreenzonearoundthecolonyonABTSplateat25?

Conday6ofincubation（Figure1a&

b）.Theligninolyticefficiencywasdeterminedmaximumat4?

Cthatdecreasedupto25?

Candagainincreasedat35?

C（Figure2）.

　　2.2LaccaseProductionAndItsMolecularMass

　　ThefungusproducedlaccasethroughoutitsgrowthtemperatureinKirk&

Farrell（1987）medium，showingpreferenceforlowtemperature.Theoptimumproductionoflaccasewasrecordedat25?

CandpH5，coincidingwiththeoptimalgrowthrequirements.Themolecularmassofthelaccasewasdeterminedtobe35kDa（Figure3）.Besides，alightbandof100kDawasalsoobservedthatmaybeattributedtotheproductionofisozymeorsomeotherrelatedlignindegradingenzyme.

　　2.3InfluenceOfLowMolecularWeightOrganicSolventsOnLaccaseProduction

　　Supplementationoflowmolecularweightorganicsolventsinthemediumresultedinvaryingeffectsonlaccaseproduction.Further，theconcentrationofthesolventsandtheincubationtimealsoinflue