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Inducing

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Inducing

    Inducing

    Chin.J.Chem.Eng.,15(4)573578(2007)

    InducingExpressionandReactionCharacteristicofNitrileHydratase

    fromRhodococcussp.SHZ.1

    1INTRoDUCTIoN

    AcrylamidefAM),acommercialchemicalwidely usedtoproducepolymersinindustrialprocessesof sewagetreatment,petroleumrecovery,papermaking andtextilesizing,ismanufacturedfromacrylonitrile (AN)mainlybychemicalprocesses1,21.

    NitrilehydrataserNHase)producedbybacteria andfungicatalyzestheconversionofalargenumber ofchemicallydiversenitrilesintoamidesandacids. TheexpressionofNHasebymicroorganismshasal

    readYbeenusedfortheindustrialproductionofAM andnicotinamide.NHasecanalsobeusedefficiently inenvironmentalremediationbyconvertingnitrile wastesintolesstoxicamides3,41.

    Greatprogresseshavebeenachievedforyearsof effortinthescreeningandoptimizationofwildstrains Furthermore,somespeciesofthewildstrainshave alreadybeensuccessfullyappliedintheindustrial productionofAMfromAN[5,61.Recently,ithasbeen givenmoreattentiontoincreasingtheactivityand improvingthestabilityofNHaseinindustrial biotransformations[7].

    Inthisarticle.thestrainRhodococcussp.SHZ1

wasusedforAMproductionbymeansofANhydra

    tion.ThisstrainisparticularlyinterestedinAMpro

    ductionsincethehydratasesystemexhibitshigh NHaseactivityandapooramidaseactivity.Accord

    ingly.verylittleamountoftheAMwasfurthertrans

    formedintotheundesiredacrylicacidinthecourseof thereaction.Inourpreviousstudy,NHasefrom Rhodococcussp.SHZ1wasproducedinthelatesta

    tionaryphaseabout8084hofthefermentation.

    whichwasharvestedatthemaximumNHaseactivity peak,ashighas942U?mgthespecificactivityfSA), insmallscalereactors[8].Inordertoimprovethe NHaseactivityfromRhodococcussp.SHZ1.thead

    ditionofinducerwasconsidered,andthereaction characteristicsoftheNHasewasalsoinvestigated. 2MATERIALSANDMIETHoDS

    2.1Strainandcultureconditions

    Rhodococcussp.SHZwasscreenedunderthesa

    lineandalkalineenvironmentfromXinJiang.And basedontheRhodococcussp.SHZ,theRhodococcus .SHZ1strainwasrebuiltintheextremelydirec

    tionalcultivation.Thesestrainswereconservedbythe Lab.ofGreenChemicalTechnologyinShiheziUni

    versity.ThecolonyofRhodococcussp.SHZ1was

    orangeandhadglossysurface.50mlnutrientbroth culturewasusedtoinoculate250mldamperflasks. Thecompositionofthemediumwasasfollows:20g?L 0fGlucose,5g?Lofyeastextract,2g?LNaCl,

    0.5g'LKH2PO4,0.5g'LK2HPO4'3H20,0.2g'L MgSO4?7H2O,18g?Lofagar.ThepHofmedium

    wasadjustedto7.2.Themediumwassterilizedby autoclavingat121?for15min[9,101.Inoculation

    preparationofRhodococcussp.SHZ1wasperformed

    inmediumwithoutaddingagar.Atiercultureat30?.

    200r.minfor48h.10%ofinoculationwastrans

    ferredto1000m1fermentationflasks.A11thechemi

    calswerecommerciallyavailablereagentsofanalyti

    calgrade.

    2.2Determinafionofbiomass

    Thebiomasswasrapidlyestimatedbyoptical densitymeasurementat600nmwavelength.Drycell massweredeterminedbydryingthewetcellsuntila Received20060326,accepted20070528.

    SupportedbytheNationalNaturalScienceFoundmionofChina(No.20466002),theProgra

    mforNewCenturyExcellentTal

    entsinUniversity(NCET-04

    089)andtheKeyResearchProjectsintheUygurAutonomousRegionofXinjiang(No.20033

    2109).

    Towhomcorrespondenceshouldbeaddressed.Email:fichun@tsinghua.org.cn 一一?一一一一一一一一一—一

574Chin.J.Ch.E.(Vo1.15,No.4)

    constantdrymass(at80.C)1l1.Fromthecalibration

    curve,oneOD600unitofRhodococcussp.SHZ1sus

    pensi'oncorrespondsto0.188mg?m1.Zeroabsorb

    ancewassetusingdistilledwater.A11theoperations werereplicatedatleasttwiceandaveragevaluesare reportedinthiswork.

    2.3InductionofNHasefromRhodococcussp.SHZ.1 InductionofNHasewasperformedbyadded

    certainamountofinducerstocultureflasksduringthe scheduledperiodoffermentation.Theconcentration ofinducerstestedwere:AN(25,50,75mmol-L), sodiumacetate(15,30,50,75mmol-L)and

    NH4CI(15,30,50,75mmol-L)solutions.These chemicalshavebeensuccessfullyusedasinducersin otherstudiesl2l51.Theflaskcultureswerestarted with10%ofinoculationbiomassconcentration.The SAOfNHasewasmeasuredevery4hduringthecul

    tivation.Thecontrolwasatreatmentwithoutaddition ofinducer.

    2.4ReactioncharacteristicofNHasefromod- OCOCCU$sp.SHZ.1

    TheactivityofNHasewasinvestigatedusing 4.75mgofcellscollectedfromtheculturebrothatthe stationaryphaseandtreatedwith200mmol?LAN, whichindicatednegligiblesubstrateinhibition.The effectofpHonreactionratewasinvestigthe ,

    atedin

    rangeofPH4.9.6.with25mmol-L'citratebuffer

    (pH4.05.6),25mmol-LNa2HPO4/NaH2PO4(pH

    6.07.6)and25mmol-LK2HPO4/KH2PO4buffer

    (pH8.9.21.200r.minand30?wereselectedto

    minimizedeactivationcausedbyshearstressand temperature.TheeffectOftemperatureontheNHase activitywasstudiedinthereactiontemperaturevaried from5to50?.Theeffectofsubstrateconcentration ontheNHaseactivitywasstudiedinANsolution,at

concentrationfrom10tol00g-L.whichwaspre

    paredwith25mmol-LK'HPO4/KH2PO4buffer(DH 7.21.InhibitionstudiesonNHaseactivitywerecarried outbypreincubatingrestingcellswithinitialAMcon

    centrationsbetween1000g-L1.Atpredetermined timeintervals(20min)asamplewaswithdrawnand washedthreetimeswith25mmol?LK2HPO4/KH2PO4 buffer(DH7.2),thenNHaseactivitywasana-

    11,16,171. lyzed

    2.5Restingcellpreparation

    Cellswereharvestedatoptimalfermentation times.Thebrothwascentrifuged.at12000r.minfor 10minatroomtemperature,andseparatedcellswere washedthreetimesusingl8mlof25mmol?LK2HPO4/ KH2POdbufferwithpH7.2.Thecellpastewasthen suspendedinthesamebufferandkeptinarefrigerator f4?)fortherestreaction.

    2.6NHaseactivityassay

    Oneunit(U)ofNHaseactivitywasdefinedas theamountofrestingcellsthatcatalyzedtheforma

    tionof1~tmolofAMperminuteundertheadopted August,2007

    conditions.SpecificactivityfSA)wasdenotedas U?mgl8,l91.NHaseactivityofrestingcellswas measuredat30?for5minbydirectassay.Thereac

    tionmixturewascomposedoflm1of200mmol?L ANassubstrate.1mlrestingcellsandl8mlof 25mmol-LK2HPO4/KH2PO4bufferwithpH7.2. Afterincubationat30?withstirredcontinuouslyat

200r.minfor5min,thereactionwashaltedbyadd

    in?lmlof4mol-LHC1.Thenthereactionmixture wascentrifugedatl2000r.minforl0min.andthe supernatantwasusedforthedeterminationofproduct andresidualsubstrate.TheconcentrationofANand AMinthereactionsystemweremeasuredbygas chromatography(GC2010,SHIMADZU),witha

    2mX4mmcolumnf5%AdvanceDS).Thetempera

    tureofcolumn,injectorandflameionizationdetector werel70?.200?,200?,respectively,andtheflow

    rateofnitrogengaswas20ml?n_..

    3RESULTSANDDISCUSSION

    3.1InducingexpressionofNHasefromodococ- cu$sp.SHZ-1

    ItwasknownthatbiomassandNHaseactivity wererelatedtodifferentinducersandtheirconcentra

    tions.TheeffectOfdifferentinducersonenzymeex

    pressionwasshowninFig.1.ThehighestNHaseac

    tivityinthecellsofRhodococcussp.SHZ1wasob

    tainedbyinducingofNH4ClandANwhenNH4Cl (75mmol?L,wasaddedintomediumatthebegin

    ningoffermentationandAN(50mmol-L)was addedafter48hofcultivation.SAandtotalenzyme activityoftreatmentwi

    ,

    thinducerwereincreasedob

    viouslytol358U-mg(Fig.2)and82l0U-ml,re

    spectively.Accordingtowhathadbeenreported,SA fromthisstrainwasthehighestone[8,201.Moreover,

cellscouldbeharvestedatthemaximumenzymeac

    tivity(6Oh).anditwassavedabout2024hcom-

    paredwiththecontrol(Fig.21.

    controlNHClCHC0ONaANNHdCl+AN

    inducer

    Figure1Theeffectofdirentinducerson enzymeproduction,

    (Horizontalbarindicatesthetypeofinducers:75mmol'L

    NH4Cl,30mmol?LCHC0ONa,50mmol?L-.AN, 75mmol?LNH4Cland50mmol-LAN.Errorsbar representsSAof^odococcus.SHZ1.Thecontrol

    wasatreatmentwithoutadditionofinducer) OOOOO?加???

    ..

    ?g.lI^ll.o?日}{Z.uo

InducingExpressionandReactionCharacteristicofNitrileHydratasefromRhodococcussp.

    SHZ-1575

    400

    200

    000

    800

    600

    24324048566472

    fermentationtime,h

    Figure2BiomassandSAcurveofRhodococcussp.SHZ.1

    inshakeflasks(1000m1)induced?L.by75mmol

    NH4Cland50mmol?L-1AN

    .specificNHaseactivity;biomassconcentration

EffectsonNHaseactivityandbiomassaccumu

    lationwerealsoobservedbyaddingNH4ClorAN separately.After72hincubation.biomassreached 6.22mg?rnlwithNrkC1f75mmol?L1and6.35mg?rnl withAN(50mmo1?L),respectively,andSAofNHase was1024U-mg_.withNHdC1.andll36U?mg_.with AN,higherthfinthatofthecontrol(942U-mg1 (Fig.11.However.ittooklongertimetoreachthe maximumenzymeactivitycomparingwithadditionOf NH4ClandAN.Otherwise,sodiumacetatehadnot anyeffectontheNHaseactivityunderchosencon

    centrations.

    ItwasclearfFigs.1and21thatadditionOfAN andNH4Clintothemediumwerenecessaryforthe highexpressionOfNHase.wheretheSAwasin

    creasedOf44%comparedwiththecontro1.According toreported.ANhasmodestlyeffectiveoninducing aneuploidychromosomeloss,andhasastructural featurethatthecyanogroupCNisadjacenttoadou. blybondedcarbonatom=C---CN13,21,22].Amide

    mightdevoteintothepuckeringactivitysiteand keepingstabilizationoftheNHase.Therefore,the betteref_fectOfNHaseactivityfromRhodococcussp. SHZ1couldbemainlyattributedtocooperationof ANandNH4Clinthisresearch.

    3.2EffectofPHonenzymeactivity

    ApreviousstudyshowedthatthechangeOf NHaseactivitywhilethePHrangingfrom6.0to8.0 wasalmostnegligible[231.Thepresentinvestigation aimedtoverifytheeffectOfPHontheactivityOf

NHasefromRhodococcussp.SHZ1.

    TheeffectofPHonreactionratewasshowedin Fig.3.Theresultsindicatedthatthehighestspecific reactionratewasobtainedatPH7.2,andtheratewas slig|ItlyaffectedintherangingofPHfrom6.4to7.6. Beyondthisrange,theNHaseactivitydecreaseddras

    tically.witl1NHaseactivityneartonilatPH4.0.It

    wasconcludedthatthisstraincouldbeusedtocata

    lyzehydrationreactioninarelativelybroadPHranges between6.4and7.6.ThebioconversionofANwith NHasefromRhodococcussp.SHZ1couldallowcer-

    tainPHfluctuationwithoutlossofperformanceduring finindustrialprocess.Thiswasratherimportantsince exactpHcontrolwasdifficultandmoreexpensivein pH

    Figure3TheeffectofpHontheactivityof

    NHaseinrestingcells

    (Runsat30?.4.75mgdrycellsand200mmol?LANin

    25mmol?L-.citrate,Na2HPO4/NaH2PO4and

    K2HPO4]KH2PO4buffer)

    theindustrialscale,whencostlywouldbetheequip

    mentsneedtokeeppHwithinanarrowrange.

    3.3Effectoftemperatureonenzymeactivity Thekineticparametersandtheactivationenergy OfANconversiontoAMweredetermined.Fig.4dis

    playedtheeffectoftemperatureontheactivityof NHasefromRhodococcussp.SHZ1restingcel1.The

    specificreactionrate,V,wasevaluatedfromplotsof productconcentrationagainsttemperature.Herethe valuesvariedwithtemperatureaccordingtothe

knownArrheniusequation[22--251:

    Vxp

    (]?

    whereAisthepreexponentialfactor.Eistheactiva

    tionenergY,Risthegasconstant,andTistheabsolute temperature.Fig.5indicatedtheArrheniusplotofAN hydratationcatalyzedbyNHasefromRhodococcussp. SHZ-1restingcel1.AnapparentactivationenergYof roughly90.2kJ?molwasevaluatedinthetempera

    turerangefrom5to30?.

    temperature.?

    Figure4Theeffectoftemperatureonthespecific reactionrateofNHaseinrestingcells

    ,(Runningwith4.725mgdrycellsand200mmol?LANin 25mmol?LK2HPO4/KH2PO4buffer,pH7-2)

    Accordingtothecurveofresidualactivityunder differenttemperatures(between20?and50?),

    catalyzingreactionbyNHaseinrestingcellsexhibited afirstorderdeactivationmechanism[26describedby

    rA=Vexp(-kdt)

    ThefirstorderdeactivationconstantofNHase, Chin.J.Ch.E.15(4)573(2007)

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