Purification of an extracellular B-glucosidase from a strain of Trichoderma reesei containing multiple bgl1 genes

155 Pages·2000·4.9 MB·English

by  Tseung, Chi-Wah

PURIFICATIONOFANEXTRACELLULARp-GLUCOSIDASEFROMASTRAINOF
TrichodermareeseiCONTAININGMULTIPLEbgllGENES
By
CHI-WAHTSEUNG
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFIMENT
OFTHEREQUIREMENTSFORTHEDEGREEOFDOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
2000
Totheladiesinmylife,
WeiYeaandEmily,yougiveme
serenityinthemidstofchaos.
ACKNOWLEDGEMENTS
I wish to express my sincere appreciation to the members ofmy graduate
committee,Dr.RobertBates,Dr.MartyMarshall,Dr.RachelShiremanandDr.Chris
West,fortheiradviceandassistanceinthisresearchwork. Dr.TimFowler,whohas
beenanactivesponsorofthisworkthroughouttheseyearsandgavemethenecessary
toolsandmeanstofinishthiswork; alsomy genuineappreciation goestoDr. Lina
Gritzaliforhermotivationandaffection.Dr.JessGregoryandhislabwereasortof
convenientstoreacrossthehallway;Icherishthehelpandsupportgiventomeduring
theselongyears. IthankthestaffmembersoftheDepartmentofFoodScienceand
HumanNutritionfortheassistanceduringmystudiesandthemarvelousgraphicworkof
Walterandhispricelesshelp.
Ithankthefollowingfriends. Sharon,Min-Ming,Rena,Eduardo,JuanPablo,
Geraldine,Maribe,youguysarespecialandmanyotherswhohavebeenwithmeinthis
experiencemyspecialgratitude. IthinkKatalin,whereversheis,formakingthetedious
timesinthelaboratorymoreenjoyableandbeingmyconfidantinthesadmomentsofmy
life.
IthankWei-Yea,mylovelywife,forbeingapermanentguidinglightbymyside
in good and bad times and her family for trusting me from the beginning always
optimisticandencouraging. IthanklittleEmilywhoinhershortlifehasgivenmea
senseoflivingandmademetreasureeveryminuteofmine. Ithankmyparentsfortheir
patienceandcomprehension;ithasbeenalongwaitingperiod.
Finallymydeepestthanksareextendedtothemostimportantand influential
personofthisdissertation,thechairmanofmycommittee,Dr. RossD. Brown. His
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knowledgeoverwhelmedmefromthemomentIsteppedintothelab. Iamgratefulfor
notonlyhisscientificguidanceandfinancialsupportduringmygraduatestudybutalso
hisqualityasahumanbeingandhisvastwisdom.Iwillmissthoselongconversations
andadvicethathasmademeabetterindividual;thanksagainRoss.
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TABLEOFCONTENTS
Page
ACKNOWLEDGEMENTS i»
LISTOFTABLES vii
LISTOFFIGURES viii
LISTOFABBREVIATIONS x
ABSTRACT xi
INTRODUCTION 1
REVIEWOFTHELITERATURE 4
OccurrenceofMicrobialCellulaseEnzyme 5
FungalCellulaseSystem 6
MeasurementofCellulaseActivity 10
InductionandRegulationofCellulaseProduction 11
SynergisticInteractionamongCellulaseComponents 16
(3-Glucosidase 18
HomologousAminoAcidSequencesofGlycosylHydrolases 19
MechanismofCatalysisof(5-Glucosidase 21
FunctionsofP-Glucosidase 23
GeneticManipulationsT.reeseiofP-Glucosidase1 25
ProductionofPolyclonalAntiseraforAffinityPurificationof
P-Glucosidase 28
MATERIALSANDMETHODS 32
PurificationofP-glucosidase 32
EnzymeActivityDetermination 33
ProteinQuantificationandPurityDetermination 34
IsoelectricFocusing 35
AminoAcidAnalysis 35
EffectofpHonEnzymeStability 36
V
DeterminationofpHOptimum ib
DeterminationofInhibitionConstants
ChickenImmunizationandAntibodyPurification 37
DuckImmunizationandAntibodyPurification 39
RabbitImmunizationandAntibodyPurification 40
MolecularWeightDeterminationofEnzymeand
Anti-P-glucosidaseAntibodies 41
AntibodyTiterDeterminationbyEnzyme-linked
immunosorbentAssay(ELISA) 42
Immunoblotting 43
N-hydroxysuccinimidylChloroformateActivatedAgaroseAffinity
Chromatography 44
RESULTSANDDISCUSSION 46
PurificationandCharacterizationofP-glucosidase 46
DEAE-SephadexChromatography(SuccinateBuffer) 47
DEAE-SephadexChromatography(ImidazoleBuffer) 47
SP-SephadexChromatography 53
ElectrophoresisEvidenceforthePurityofP-glucosidase 56
IsoelectricPointofP-glucosidase 60
CompositionofP-glucosidase 61
RelationshipofpHtoP-glucosidaseStability 63
OptimumpHforP-glucosidaseActivity 67
DeterminationofKineticsandInhibitionConstantsforthe
PurifiedP-glucosidase 74
ProductionofAntibodies,ImmobilizationandAffinityPurification
ofP-glucosidase 91
ChickenAntibodyProductionandPurification 92
DuckAntibodyProductionandPurification 98
RabbitAntibodyProductionandPurification 99
AntibodyTiterDetermination 104
ImmunoblottingofMultipleAntiT.reeseiP-glucosidase
Antibodies 104
InhibitoryEffectofChickenAntibodyonP-glucosidase 107
AffinityChromatographyPurificationofP-glucosidase 117
SUMMARYANDCONCLUSIONS 123
REFERENCES 128
BIOGRAPHICALSKETCH 139
vi
LISTOFTABLES
Table Page
1 EnzymescomprisingthecellulasesystemofTrichodermareesei... 8
2 Assayproceduresandsubstratesusedforcellulaseactivity
measurements 12
3 PurificationsummaryofP-glucosidase 57
4 AminoacidcompositionofP-glucosidase 62
5 KineticandinhibitionconstantsofP-glucosidase 87
vii
LISTOFFIGURES
Figure Page
1 Cooperativeactionofenzymesofthecellulasesystemin
hydrolyticallyconvertingcellulosetoglucose 9
2 ElutionprofilefromDEAE-Sephadexcolumnchromatography
(succinatebuffer)ofdialyzedculturefiltratesofTrichodermareesei
containingmultiplebgl1genes 48
3 ElutionprofilefromDEAE-Sephadexcolumnchromatography
(imidazolebuffer)ofcombinedfractionsenrichedinP-glucosidase
activityelutedfromthesuccinatebufferDEAE-Sephadexcolumn 52
4 ElutionprofilefromSP-Sephadexcolumnchromatographyofcombined
fractions10-13elutedfromtheimidazolebufferDEAE-Sephadex
column 54
5 Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis(SDS-PAGE,
7.5%gel)profileofpurifiedP-glucosidasefromdialyzedculturefiltrate
ofTrichodermareesei. 58
6 Effectofhydrogenionconcentrationonthestabilityofpurified
P-glucosidase 65
7 Dixon-Webblogplots:LogVvs.pHandlogV/Kmvs.pH 68
8 OptimunpHofpurifiedp-glucosidase 72
9 Lineweaver-BurkdoublereciprocalplotforpurifiedP-glucosidase 75
10 Lineweaver-BurkplotofP-glucosidaseinhibitionbyD-glucose
using/j-nitrophenyl-P-D-glucopyranosideassubstrate 78
11 DixonplotofP-glucosidaseinhibitionbyD-glucoseusing
/Mutrophenyl-P-D-glucopyranosideassubstrate 80
viii
12 Lineweaver-BurkplotofP-glucosidaseinhibitionby
1-deoxynojirimycinusingjp-nitrophenyl-P-D-glucopyranoside
assubstrate 83
13 Dixonplotofp-glucosidaseinhibitionby1-deoxynojirimycinusing
/Miitrophenyl-P-D-glucopyranosideassubstrate 85
14 DixonreplotofinhibitionofP-glucosidasebyD-glucoseand
1-deoxynojirimycin 89
15 Profileofanti-P-glucosidaseantibodyproductionineggyolksfrom
immunizedhensduringtheimmunizationperiod 93
16 Sodiumdodecylsulfatepolyacrylamidegelprofileofchickenanti-P-
glucosidaseantibodypurifiedbyPEGprecipitationandPhenyl
SepharoseCL-4Bchromatographycolumn 96
17 Sodiumdodecylsulfatepolyacrylamidegelprofileofduckanti-P-
glucosidaseantibodypurifiedbyProteinASepharoseCL-4B
chromatographycolumn 100
18 Sodiumdodecylsulfatepolyacrylamidegelprofileofrabbitanti-P-
glucosidaseantibodypurifiedbyHiTrapProteinGchromatography
column 102
19 Titerdeterminationofantibodies(chicken,duckandrabbit)against
P-glucosidase 105
20 Specificitydeterminationofanti-P-glucosidaseantibodiesby
immunoblottingtechnique 108
21 Lineweaver-BurkplotofP-glucosidaseinhibitionbychicken
anti-P-glucosidaseantibodyusing/?-nitrophenyl-P-D-glucopyranoside
assubstrate Ill
22 DixonplotofP-glucosidaseinhibitionbychickenanti-P-glucosidase
antibodyusing/?-nitrophenyl-P-D-glucopyranosideassubstrate 113
23 DixonreplotofinhibitionofP-glucosidasebychicken
anti-P-glucosidaseantibody 115
24 Sodiumdodecylsulfatepolyacrylamidegelprofileofpurified
P-glucosidasebyduckhydroxysuccinimidyl-agaroseaffinity
chromatographycolumn 119
ix
Sodiumdodecylsulfatepolyacrylamidegelprofileofpurified
P-glucosidasebyrabbithydroxysuccinimidyl-agaroseaffinity
chromatographycolumn