bioprocess practical 1.docx
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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
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