bioprocess practical 1.docx

bioprocess practical 1.docx

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bioprocesspractical1

Title：

ENZYMEIMMOBILIZATION:

ImmobilizationofEnzymesbyGelEntrapment

Name：

ZhangXiaoyun

X00097360

Nameofpartner：

MojiOlafisoye

ZhangHaoran

Date：

24/09/2014

Abstract:

Enzymeimmobilizationhasbeendevelopedfordecadesandbecomeanimportantpartofindustryandlab.Theexperimentisabouttheimmobilizationbygelentrapment.Theexperimentdeterminesagoodworkingdilutionofenzymefirstly，andthencomparetheactivityandspecificactivityofenzyme（horseradishperoxidase）inCa-alginategelandPolyacrylamideacidandthesolubleenzyme.

Introduction

Solubleenzymehasbeenusedforfoodindustryforhundredyearsandhasapplicationsinthepharmaceuticalandchemicalindustriesinrecentyears.Theirstructureandmodeofactionaregraduallybeingelucidatedthroughavarietyofwell-knownexperimentalmeans,suchasX-rayandNMR.Moderngeneticengineeringmethodshavebeenusedtoproducealargenumberofenzymesandmodifytheirprimarystructuretochangetheirphysic-chemicalandbiologicalpropertiestoletthatsomeoftheenzymeismoresuitableforindustrialproduction.

TheresearchofImmobilizedenzymebeganin1910andhadbeencarriedoutaroundtheworldsince1970.Immobilizationisthetechnologythatletenzymeboundwithsolidmaterialsorlimitedtoacertainareaandenzymecanstillperformitscatalyticreactionanditisrecyclableandreusable.Comparedwiththefreeenzyme,immobilizedenzymecannotonlymaintainitshighspecificityandmildreaction,butalsoovercometheshortcomingsofthefreeenzyme.Itismorestableandcanbeseparatedandrecoveredeasierthanfreeenzyme.Immobilizedenzymehasbeenappliedpopularlynotonlyinthefieldsofchemistry,biologyandbiologicalengineering,medicalandlifesciences,butalsomeettherequirementsofsustainabledevelopmentstrategybecauseitsavesresourcesandenergytoreduceorpreventpollution.

Themainadvantageoftheimmobilizedbiocatalystiseasytocontrolthereactionandtheproducteasilyrecoveredandpurifiedandhaveawideselectionofreactor.Butitalsohasthefollowingdisadvantages:

activityisreduced.Substratesofreactionanddiffusionofproductsarelimitedandthecostincreases.

Thetechnologyofimmobilizedenzymesintheindustryhasdevelopedrapidlyandhasincreasinglybecomeamatterofrationaldesign.Numerousmethodsofimmobilizationonavarietyofdifferentmaterialshavebeendeveloped.Theycanbeclassifiedintotwomaincategories:

PhysicalandChemicalmethods

Physicalmethods:

1.Absorption2.Entrapment

AdvantagesofPhysicalmethods:

theenzymedoesnotparticipateinchemicalreactionssotheoverallstructureremainsandthecatalyticactivityoftheenzymeiswellpreserved.However,becausetheembeddedorsemi-permeablemembranewithacertainspaceandthehindranceeffect,itcannotbeappliedforsomereaction.

1.Absorptionmethod:

Anenzymeorcellcontainingenzymeareabsorbedonthesurfaceofthematerials.

2.Thecarrierbindingmethod:

itismorecommonusedthantheAbsorptionmethod,

Chemicalmethods:

1.Combiningmethod（includingionicbindingandcovalentbinding）,crossing-linkmethod.

Itconnectstheenzymeandthenaturalorsyntheticpolymercarrierbyachemicalbond.

Therearediverseformsofimmobilizedenzymewhichcanbemadeintoparticleswithgoodmechanicalpropertiesforcontinuousproductionorhaveabatchstirredreactioninareactor.Itcanalsobemadeintoenzymemembraneandenzymetubesusedinanalyticalchemistry.Microcapsuleswithenzymecanbeusedasatreatmentforclinicalapplication.Nowadays,peoplecancombineenzymemembrane（includingmembraneofcells,tissues,microorganism）andelectricalsensitivecomponents（light,heat）intoabiosensorforthedeterminationoforganiccompoundsandharmfulsubstancesinenvironment.

Immobilizedenzymehasmorefeaturesthansolubleenzymesolution,butitwaspreparedtogothroughalotofcomplexseparationandpurificationoftheenzyme.Onekindofimmobilizedenzymecanonlybeusedforspecificsingle-stepreaction.Somoreandmoreindustrialcompanystartedtousetheimmobilizedcelltechnology.Itobviatesthetimeofseparationandpurificationoftheenzymeandsimultaneousmulti-enzymereaction.Enzymeinthecellcankeeptheoriginalform,increasingthestabilityofenzyme.

MaterialsandMethods

Reagents:

1.Horseradishperoxidasestockenzymesolutionat1.0mg.ml-1inHEPESbuffer

2.Peroxidasesubstratesolution:

dissolve10mgphenolin40mldH2O;

add25mgof4-amino-antipyrine;

dilutetoafinalvolumeof50mlwithdH2O

3.H2O2solution（preparefresh:

1.7mM）→usedtostartthereaction:

mix1mlof30%with99mlofdH2O;

dilute1mlofthissolutionto50mlwith0.2MHEPESbuffer

4.2%w/vSodiumalginate（Alginicacid-sodiumsalt）

5.Ammoniumpersulfatesolution（fresh;0.1g/mlin0.2Mphosphatebuffer）

6.KOHsolution

7.Stockacrylamidesolution–40%w/vacrylamide

8.Stockbis-acrylamidesolution–2%w/vofmethylenebis-acrylamide

9.HCLsolution

10.0.2MPhosphatebuffer,PH7.2

11.0.03MCaCl2

12.TEMED

13.HEPESbuffer,0.2MPH7.2containing0.2MCaCl2

14.0.2MCaCl2

Method

3.1DeterminationofaWorkingDilutionofHorseradishPeroxidase

Preparationofblankandusedblanktozeroaspectrophotometer

Blankcontained:

0,5mldH2O,1.0mlofaminoantipyrine-phenolsubstratereagentand1.0mlH2O2andwaitedfor2minstomakesurenoreactioninit.

Preparationofthedifferentdilutionofenzyme（1:

25,1:

50,1:

100）

Dilution

Enzyme

dH2O

1:

25

0.04ml（40ul）

0.96ml（960ul）

1:

50

0.02ml（20ul）

0.98ml（980ul）

1:

100

0.01ml（10ul）

0.99ml（990ul）

Inacleantesttubeshouldcontain:

0.5mldilutedenzyme,1.0mlaminoantipyrine-phenolsubstratereagentand1.0mlH2O2（finallyaddedtostartthereaction）

Placethosethreedilutionofenzymeintospectrophotometerrecordtheextinctionat510nmat30sintervalsover5mins.

Haveagraphofeachenzymedilution.

3.2MeasurementofHorseradishPeroxidaseActivityandSpecificActivity

Blank:

0.5mlofdH2O,1.0mlsubstratereagentand1.0mlH2O2

Selectedthebestworkingdilutionofenzyme（1：

100）anddotheenzymeassay

Inacleancuvette:

Addedtheenzymeand1.0mlaminoantipyrine-phenolsubstratereagent

Finallyadded1.0mlH2O2tostartthereaction.

Differencewithmanual:

Manual:

Inacleancuvette:

1.0mlaminoantipyrine-phenolsubstratereagentand1.0mlH2O2

Finallyaddedthe0.5mlofthedilutedenzymetostartthereaction.

Allowedthereactiontoproceedfor3minutesexactly.Recordedtheextinctionat510nm（A510nm）onthe3minmark.Thereactionshouldbeperformedintriplicate（1blank2assay）.

Calculatedtheenzymeactivitypermlofneatenzymeandthespecificactivity.

3.3ImmobilizationofHorseradishPeroxidaseinPolyacrylamideGel.

Didn’tdothe3.3

3.4PreparationodBlankCa-AlginateGelBeads

Mixed10mlof2%w/vNa-alginateand5mlof0.2MHEPESbufferwell.Setupabeakercontaining:

40mlof0.2MCaCl2andStircontinuously.Puttheburetteoverthebeaker.DroppedthesolutionintotheCaCl2solution.

Allowedthebeadstocurefor15minsandthenwashedwith0.03MCaCl2solution.

3.5ImmobilizationofHorseradishPeroxidaseinCa-AlginateGel

Mixed4mlof2%w/vNa-alginateand2mlofhorseradishperoxidase（1mg.ml-1inHEPESbuffer）

Transferredthemintoaburetteonaretortstand.Droppedthesolutionintoastirredbeakercontainedof20mlof0.2MCaCl2andstirredcontinuously.Allowedthebeadstocurefor15minsandthenwashedwith0.03MCaCl2solution.

Weighthebeadswithenzymeandwithnoenzyme.

3.6EnzymeProgressCurvesforSolubleandImmobilizedHorseradishPeroxidase

Solubleenzyme：

theenzymeactivitycurvesdevelopedundermethod3.1

Immobilizedenzyme:

Firstly,Setupablankreaction:

Toauniversaltube,added0.5gofgelwithoutenzymeand2.5mlofaminoantipyrine-phenolsubstratereagent.Finallyadded2.5mlH2O2（time0）

Mixedwell,thetubeshouldbemixedgentlyandcontinuouslyduringtheassayperiod.

Checkedtheabsorbanceagainat3minutes.

Usethetubeto‘zero’yourspectrophotometer.

Secondly,Prepared0.25ggel

Toauniversaltube,Added0.25gofgelimmobilizedenzyme,2.5mlofaminoantipyrine-phenolsubstratereagent.Finallyadded2.5mlH2O2（time0）tostartthereaction.Mixedwell,thetubeshouldbemixedgentlyandcontinuouslyduringtheassayperiod.

Atexactly45,90,135and180seconds,recordedtheextinctionat510nm.

PlottedagraphofA510nmversustime.

Thirdly,Prepared0.5ggel

Toauniversaltube,

Added0.25gofgelimmobilizedenzyme，2.5mlofaminoantipyrine-phenolsubstratereagent.Finallyadded2.5mlH2O2（time0）tostartthereaction.Mixedwell,thetubeshouldbemixedgentlyandcontinuouslyduringtheassayperiod.

Atexactly45,90,135and180seconds,recordedtheextinctionat510nm.

PlottedagraphofA510nmversustime.

3.7AssayofImmobilizedPeroxidasetoMeasureImmobilizedEnzymeActivity

Asablankreaction:

Added0.5gofgelwithoutenzyme，2.0mlofaminoantipyrine-phenolsubstratereagentand2.0mlH2O2

Asanenzymeassay：

Added0.5gofgelwithenzyme，2.0mlofaminoantipyrine-phenolsubstratereagentand2.0mlH2O2

Allowtheassaytoproceedforalengthoftimeequivalenttothelagphase.Takethistimeastime0，andrecordedtheextinction.Mixwellandcontinuetomixgentlyandcontinuouslyduringtheassayperiod.

Atexactly3minutesaftertime0decantedthesubstratesolutionminustheimmobilizedenzymetoacleancuvetteandrecordedA510nm.

Calculatethespecificactivityoftheimmobilizedenzyme.

Results

3.1

Figure1

Time（S）

Blankdilution

1:

25dilution

1:

50dilution

1:

100dilution

0

0.000

0.178

0.068

0.029

30

0.000

0.630

0.092

0