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characterizationofthecontar. :ir
. atinbr. : :
:terialswhenthecellsareincubated
withpureglucose . InthiswaythecontributionofthehighDEsyruptothe
impuritycontentoffinalisomerizedsyrupcanbeevaluated
.
Approximately450gallonsofisomerizedsyruphasbeenpreparedin
thisperiod. Fourhundredgallons(2batches)weresenttoProductDevelop-
mentforclean-upforuseinfeedingtrialswhile50gallonsweresentto
CedarRapidsforaclean-upevaluation. Duringtheinitialstagesofthe
clean-up,ahazematerialwasobservedinthesyrupafteragranularcarbon
treatment. Furtherstudieshaveindicatedthatthehazematerialcanbe
removedbyafiltrationandmixedbedion-exchangeresinsorbyafinal
powderedcarbonmixture(20%DarcoG60carbonand80%StandardSuperCel
filteraid) .
Thesyrupforthefeedingtrialswastreatedwithgranularcarbon,a
C-25cationresin,anIRA410anionresin,andthepowderedcarbonmixture .
ThefinalsyrupdidnotequalIsomerose100inanacceleratedheatstability
test. "'--
StudiesinCedarRapidsandatProductDevelopmentdemonstratedthata
granularcarbontreatment,mixedbed(IRC120andIRA93),C-25andA-57
ion-exchangesystemproducedaglucose-fructosesyrupequaltoorbetter
thanIsomerose100intheacceleratedheatstabilitytest . Theseresults
willbeverifiedinapilotexperimentinCedarRapidsthismonth
.
Anappropriatealiquotoftheisomerizedsyruphasbeensentto
IndustrialBio-TestLaboratoriesfordogfeedingtrials
. Anotherbatchwas
senttoNutritionalBiochemicalforincorporationintoadietforratfeeding
studies. Theapproximatecompositionofthissyrupis :46%fructose,51%
glucose,<0. 1Zmaltoseand<0 . 1%isomaltose . Thesedatasuggestthatthe
granul'arcarbonandthefinalpowderedcarbonmixtureremovedthe
dissacharidesfromthesyrup
. -
BetweenAugust14andNovember14,atotalof4168sampleswereanalyzed
forisomeraseactivity,glucose,fructose,ortotalreducingsubstances .
B. BIOCHEMICALSYNTHESIS
I. GlucoseIsomerase
Themajorityofthefermentationseriesinthisperiodwereusedfor
theproductionofcellsforconversionoflargebatchesofsyrup
. Approxi-
mately450gallonswereusedforfeedingtrialsandpreliminaryclean-up
studiesand,eventually,a1,000-gallonbatchwillbeusedforclean-up
.
studies. Duetotheseproductionruns,littlefermentationtimehasbeen
availableforstudies . onincreasedyieldsofglucoseisomerase .
However,ithasbeenobservedthattheyeastextractusedinfercnenta-
tionmediacontainedheatresistantbacterialsporeswhichcontaminated
thefermentationr. ~ediumaftersterilizationinboththe30-gallonfermentors
andintheshakeflaskcultures
. Whenexcesssterilizationconditionsare
usedtoeliminatethesespores,theglucoseisomeraseactivityissubstantially
reduced . Therefore,theyeastextractissterilizedseparatelyandthenitis
addedtothebasalmediumpriortoinoculation
.Anincreascinthemagnesium
contentof25ppairesultedinanincreaseintheglucoseisomeraseactivity
ofthecells.Furtherincreasesofmagnesiumexhibitednosignificanteffect
ontheactivityofthecell
.
Inthelast24fermentationrunstheincubationtimehasbeenreduced
fromthenormai60to66hourstoanincubationtimeof47to52hours .
Duringtheserunsareductionintheenzymaticactivityofthecellshas
beenobserved
.However,thisreductiondoesn'tappeartobeduetothe
shorterincubationtime,sinceglucoseisomeraseactivitiesof700to800uU/ml
havebeenobservedin47to50hoursatthestartoftheproductionruns.'
Thereasonsforthisdropinactivityareunderinvestigationwithspecial
emphasistomediumstandardizationandculturevariability .
Four100-gallonconversionswereperformedusingunfilteredhighDE
syrupandthesecontained43to47%fructose .Acompositeofthesefourare
usedforfeedingtrialsandcontained46%fructose .
TwoconversionsusingbothfilteredandunfilteredhighDE_syrupwere
performedforclean-upstudiesbyPenickandFordpersonnel .The20-gallon
batchpreparedfroma'r,ighDEfilteredsyrupcontained46%fructose
.
However,a10-gallonbatchpreparedfromaSil-FlofilteredhighDEsyrup
onlycontained34%fructose
.Sincethereactionconditionsandenzymecon-
centrationswerethesameinbothcases,theloweredfructoseconcentration
maybearesultoffiltration
.Ithasbeenobservedthatlowconcentrations
ofiron(10-3i•i)inhibittheenzyme .Sincethisfilteraidwasnotacid
washed,someinhibitoryionscouldhavebeencarriedoverintothesyrup,
hence,reducingtheenzymaticactivityofthecellsand,therefore,reducing
thefinalfructoseconcentration.
Sinceitisdesiredtore-usethewholecells,alaboratoryconversion
wasperformedusingone-literbatchesofsyrup
.Theinitialsyrup,using
freshcells,containedapproximately35%fructoseafter12hoursand43%
after24hours
.Thecellswerethenremovedfromthesyrupbycentrifugation
andresuspendedintooneliteroffreshsyrup(equivalentto1partcellsto
1partsyrup)andinto500mlofsyrup(2
:1ratiocellstosyrup) .After
12hoursofincubation;thesyrupwiththeratioof1
:1contained16 .5%
fructosewhilethe2 :1ratiosyrupcontained26%fructose
.Thesevalues
werecorrectedforthezerotimecontrols
.At12hourstheisomerization
curveswereessentiallyflatwhichsuggestsverylittle,ifany,increasein
fructoseconcentrationuponfurtherincubation
.
Studiesontheproductionofacell-freeenzymefromArthrobacterhave
shownthattheenzymecanbereleasedbyalysisprocess
.Twoalternatives
havebeeninvestigatedtodeterminetheconditionsrequiredtoremovethe
enzymefromthecell
.Theenzymecanberemovedbytreatmentwithlysozyme
(1mgper25gofcells)andinsonication .
:Theenzymeisthenprecipitated
withequalvolu.-r~esofacetoneat-10°C
.A40%conversionofglucoseto
fructosewasobtainedwiththisenzymeprecipitatein12hours,whichis
approximatelythesameresultsobtainedwiththewholecellsystem
.However,
thecolorformationwasgreatlyreducedinthecell-freeconvertedsyrup
.
Thecell-freeenzymecanbetightlyboundtoDEAE-cellulose
.Theenzymeis
difficulttoremovewithwaterorde_•:trosesolutionsbutitcaneasilybe
removedwith0.3*isodiumchloride .Mhenanequivalentamountofenzyme

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