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(CG'fase)from Bacillassp' TS1-1:
Productionof CyclodextrinGlucanotransfcrase
Process0Ptimisation
Azmil
Haizam Ahmad Tarmizir Rosli Md. Illias2 Madihah Md' Salleh3 OsmanHassana
- -r.-"rrir"ti"t
ramaruddinj RoshanidaAbdulRahnran6 Suraini Abdul AzizT
EkgineennS'
FatuLt! afChenicoLE^gittt.riry and NaItuol Resoutces
Ur^,!rtiti TeknabBiMatutskt,8ll lA Ska.ldi,Johor Maldlsi.t.
TeL 6A-7-5535172. Fa\: 6'07'558I 463
Enn iL: hdizan1,79@la hoa. ofi
'Enoil: ftasli@ktnhtY
6Ehail: ranida@ 4.n!
rD.P|nnent oJBiata$
Facrlt! o[Scienc.,
Ltn^e rsiti TeknolagiMak 'rid,3l3IA Skdai, Johat,Mtktltid
TeI : 60-7-553$2A. Entti I : tnatli hah@kinia li. tu n ta'l
aSchaotaJCheDtit:dL
and FoodSciences,
Facult! af Scienceatld Techtnlog',
Urive$i t i KebdnllsaanMaLorsia,430U Bangi, SekutSoLMalavtia
5Bioprccess
an.I CherticaLTechMloS!Centre
SIRIMRerhad,Malalsi(t
l Facultyof Food 7'cc1"1o1os!
anl FoodScience,
Unire,si ti Putn Malaysia, 43100 Setuans, SelanSol Malavsia
Tel: 60-3-89468367.Far: 6'03'89423552
Abstract
Ihe effect d pH a^LI rcnperatutu flere iirestis.lvdfor on
optnrked condition oJ cycLodextth Slucanota tJeruse
GATdse) pra.lu.tion fron BdcilLus sp. TSI t Thc
aptimisation process |9as anaLlsed using Centtul
Canpasite ExperinlnnL Deiisn h ResPo$e Su'face
Llethodoloty (RSM) by Desi|, Eqert V.Nian 60.4
(StatEase,USA). This .Iesip was enpLoled to d.nre a
statistical tnodel for the eJIec^ of pH an.l tetnPerctrre an
the pto.luctian oJ Ccrase fron BaciLlussp. TSI'1. The
Rz was found to be 97 851o.
coe:licient o.f .letenintion,
n1e rcnlpentureof29.5q'C and te PH oJ 10.32had been
lau4d Io be ttp aptidtunt.onJ'lioht lbr th. ncLtihun
production of CGTaseaJter 21 hours of iac\bation tn these
conditiohs, the CGTase activitf wat diaine.l to be 75 46
Luhll. which \|6 clop ta tlz h1odelPrc.lictions
Keywords:
OptimisalionCondition.
CyclodextrinGlucanotmnsferase,
COTrse Acdvity, Prcduction. Response surface
Methodology
Introduction
CGTase(EC 2.4 t 19)has
CyclodextringlucanotransfeLase,
in both
beenfound in severalbacterinlspeciesand catalyses
236
of o'
iniermolecula.and int.amolecula.transglycosylation
1,,1glucan. The reaction between CGTase wilh starch
produced cyclodextrins (CDs) with variety of
malooligosaccharides
tll. CGTase is a mulliiunctional
enzyme,whercasbes;descyclization (!he conversionol
slarch and relared o-1, 4-glucansinto cDs thrcugh an
inlmmoleculartransglycosylatiotreaction) this enzyme
mana8esto catalysea coupling rcaction(openingof CD
to
rings and lmnsfering of linear mallooligosacchandes
acceptos) ihrough intermol@ular rransglycosylalron
reactions-Nevedheless,CGT$e had a week contribulion
towardshydrclysingactivitywilh watermol€culest2l.
Cyclodextr;ns(CDg have a structureof oligosaccharide
rings comprjs;ns '1-1. 4linked slucopymnosere$d!es.
Cyclodextrins are also known as cycloamylosesor
Schardinger
Dext.ins. The are mainly thr@ commontypes
of CDs. which a'€ made up of 6, 7 or 8 molecules,
identifiedas !-, ll- and u-cyclodextrinsresp4tively CD
molecules have a torueshaped structures,wherc the
hydrophiliehydroxyl groups at the exterior site and the
hydrophobicC-H sroups and glycosidic oxygen d the
ofCDs
interiorcavity.lD the recentyears.the requhements
have received grear attentions, especially in food,
pha.maceutical,chemical. cosmetic as well as the
asriculturalinduslriesi3l. The reasonbeing is dre capable
ofCDs moleculesto fomr incllsion complexeswith a wide
variety of guest molecules(organicor inorganic)withou!
changingthe propertiesof fie guesl molecule ir selves.
CDs are atsocapableto slabilizelabile materials,maskoff
ISBN:983-2643-15-5
Pr.ct.tlhSs
odous, emulsity oils and chdge
into powde..
comPounds
viscous or oily
Among the th.ee main types of CDs produced,D-CD is
rnorewidely used and developedtbr variousapplications.
Owing !o ils low solubility in water, -l-CD is readily
from lhe rcactionmixture withoul any lreatment
separated
wirh orSanicsolveDtsand ils inclusioncomplexescan be
easily prcpared. The capability of predominantly!-CD
produciDgcan reducesubseqtrerlpurificationcosts,leading
to commerc;allydesirabilily.Therefore.effofts arelbcused
on finding the suitableCGTaseproduce$for an efficientof
'CD production.
Approach arld methods
Culture ConditioN for CGTaseProduction
Ao.ill!.r sp. TSI I was grown in optimisedmediunrwith
rhe compositionof 2% ($lv) s.so starch.19. (w/v) yeast
extract. 0.1% (w/v) K,HPOa atd 0.02Eo {w/v) Mg
SOr.?HlOand addedwith l0% (w/v) ofNa,COr separately.
dependingon the pH ol fte medium. The cells were
c ltLrredin 250 ml conicrl flasks and incubaledat |he
Empe.arureof 37oC wilb the agitationof 200 rym ior 18
hours.The cellswerc thencentnf!8edat 501ruem ibr 4 to
5 minutes,walhedoncewith no.mal salinesolution(0.85'.4
Nacl), giving a lu.bidi!y (oplicaldensity)of0.5 41600nm.
About l07o of bacterialinmulum was inoculatedinlo the
medjum.giv;ng rhe total volume of 100 ml (includingthe
medium)in each500 ml conical flasks-The cultureswere
then incubated al the rotation speed of 100 rpn wilb
lemperaturc shown in Table I fo. 48 houn in an orbild
shaker.Sampleswere hanesledevery 2 houn for the fi61
24 hoursard every4 hoursfor the next 24 bou.s.ln every
samplirg, rhe sample was centrifugedat 5000 rpm for 2
minutes to separatelhe bacterial cell and the clear
CGTase Assay
The CGTas€ aclivity was measuredusing ihe method
by t4l. The .qtction nixlures containing40 mg
esrablished
of soluble starch in 1.0 ml of 0.1 M sodium phospha€
al
buffer (pH 6.0) and 0.5 ml supernatantwas ancubated
60"C fbr l0 minutes.The reactionwas stoppedby adding
3.5 ml of 30 mM NaOH, followed 0.5 ml of 0.0?% (wv)
phenolphthaleinin J mM Narcq solution-rhe colour
inFnsity of the samples was measurcd al 550 nm afler
The blank
slandingup for 15 minutesat room temperature.
solution, which lack of enzyme (m€dium) was prepared lor
eachbatchof assays.Standardcurve using0.1% v of ll-
Results
From the experimentaldesign,experimentswith different
combinationof temperaturcand pH were performed.The
CGTase activities of each s€ts of experiment were
d€termined after he cul$re was cultivat€d for 24 hours.
The equationobtainedafleranalysisof variance(ANOVA)
ISBN:981-2643-15-5
af Inxmotiona! Ca'!f.En.e Ar Ch.mi.al and BiL?taces Et$nt1titl!
27r' 2qt Aut:d?003, U,tnPt\itiilolnrsia Sabrh.Kttt Kt)atalr
CD (Fluka) to repLacesoluble strrch and c.ude enzyme
re$ecdvely was plotted. A unit of erzylre acllvrry wrs
definedas the amounlof enzymethal forms I $nol of F
Drt Cell Weight D€terminalion
The.e werc rwo approaches
in lhe cell mrss dererminltion,
dependingto lhe starch presencein the culture- li the
(Novo
culture containssta.ch.0.L ml enzynre
--anylase
Nordisk) was added1o I ml of culturc to hydrolysethe
residuestarch to soluble sugars [5]. Tfie mixlure was
incubatedat 100'C fof 20 nrinutesand centrifugedat 3000
efl for 30 minures. The supenritanlwas thcn decanled
_im
and the cel l s sere fi hered oni o prc$ei ghted0 . 2
cellulose nifiaG fiher (Wh.tmrn). $ashed twice with
distilied watef lnd dried in rn oven aI 95!C ibr 24 hours.
Freshmediumsas trextedsimilady and usedas blaik for
reductionofstarch impudticsin lhe sanrple.
Il srarchwas depletedfrorn the samplc,rhe cell masswas
nreasured
by taking I ml ofcuhure and cenr.ilugedar 5000
The cell pellel was
rpm for 3 minutesin a microcentrifuge.
washedtwice with saline solution aDd died in vacuun
drier, lollowed by drying in the oven at E0 "c to constart
the cell weight-The presence
of slarchin the cuhtrrecan bc
deleminedby usingiodinesolution.
Starch Concentration
Starch concentmtion in lhe sample was canied out
acco.dingto lbe methodof t6l. 1.0 ml of srpernatantwas
mi xedsi rh 4 ml ol 001 M i odi l e | l 025 M po r ds. iur ,
iodide (KI) and diluted wirh 15 ml of distilled waler.The
colonr intensitywas measuredal 465 nm againstblank of
distilledwatertreatedin lhe samenanner above.The starch
in culturefiltrate was quantifiedaccordinSto the slandard
curveof sttuchbelween0 to 3.5 mg/nrl.
Protein Content
Protein conrent was determ;nedaccording to Modified
LwD' PrcreinA,sa) I7l.
Exp€rimental Derign
Experimenlal design was determjned based on lhe
establisbedmethodt8lt9l. Tbe effect of temperalureand
pH were studied using Central Composite Des;en in
ResponseSurfaceMethodology(RSM). The optimisation
was performed to the total sets of l7 experiments,
consistin8four factorialpoinls,eight axial (star)pointsand
Iive cente points. Tbe ranges chosen for botr process
variableswere 9 to ll for pH and 25oC to 35'C for
remperature, The CCTase aclivily was chosen a1 the
du.ation of 24 hour in every run of experimenls.
givesthe CGTaseproduciionfrom Ba.ir6 sp. TSI-1 as a
function of lemperatu.e and pH. The final equaiion (actual
value)w6 exprcssedin secondorder polynomialequarion
0).
X= 2252.35464+51.59356A+301.62010B-0.90418A?15.05414B1
+ 0.27550A8
(l) where X=CGTde activiry, U/ml, A=Tempera re, "C
andB=pH.
237
Pnaalklt
The rhree dinreisional presentation(co.relxtionberween
reDpemrure
rnd pll on rhe ccTase production)lvasshown
in Figure l. The optimnm temperatureand pH lbr lhe
maximun CGT.se production werc 29.59'C and 10.32
respecrively,giving ! prcdicted CGTrse productionof
7 6 .17U/ m l.
The op{inru|rconditionsfor both rempenturernd pH were
applied in the sh.ke ilasks to conrpare lhe CGTase
p,!duction with the predictedor€s. BeskLes,lhe grcwth
kinctics of the nicrobc was also studied.The CCTase
prcducrionat 24 houlSincubationwas oblainedIo be 75.48
Ll/,nt. whi.h $rs closedto the nrodelhad p'tdicted. The
CGTaseproducrioniourrl ro be consrantatter l8 bou6 of
Discussion
The CGT.se production.s a functionof lemperalureand
pH is $rosn in equntion(l). The fitnessof the nrodelrvas
expessedby rhe coefilcienrof determination,Rr. The R2
value, 0.9785 oL 97.85'Z indicate the !a.iability in the
.l httltti.krl
27' 2q
Ca'*ratu
On ChentetL tnd Biopn..s
Erlittitt
Arfui 2AA3. Urliftrxiti M.la,"sh Sahah Kotu Ktnnnh
rcsponseof the model, whe.e 2-15610
of lhe total varirtion
was not explainedby tlre model(equation-l).The valueof
adiusleddeteflninationcoefticient(Adi R' = 0.9687)was
found to be hish lnd .dvocatedfor a hish sisnificanceof
1bemodel.The coirelationcoefficient.R lvas mai.ly used
to show the corelation measu.ement
of estimationin lhe
regression
model.The closerthe R !.lue to the unity, the
beuerco.Lelationbetweenthe obseNedand the predicted
valueswas gain. From ANOVA, the R valuewas found !o
be0.98,12.
As mentjonedpfeviously, the oplimum conditions ibr
mllximum CCTrse prcduction lvere pH 10.12 and
lempemtureof 29.6"C,giving n predicrodvalue ot 76.17
U/ml. From lhe rwo-dimensionalpr€seirarion of dre
rcsponse
sudace,it was observeddratthe optlnum CGTase
productionresponsewas more sensitiveto chrngesin one
pH unit rathertban one temperaturennit (t). Thereib.e.
lhe responsesu.frce analysisof Lheexperimentrlrcsulrs
cleirly revealsrhe intenelationshipbelveen reinperarure
andpH asafecting theCGTaseprodlction.
764m7
642494
CGTase
Act. 52.oso1
399303
277715
Figure I : The lhrce-dnnensio'1alpresentatian of the rcspansesurfacefor th. CGTdseauiyirJ of Bacillus sp. TSI 1. Tenperdture
units are in.legrees Celcirswhile the CGTaseactivit! unns werc i1tU/nl.
The lemp€ratureaboveo. below lhe oplimum temperature
(29 6 C) was observedro give low CGTaseproduclion.A1
lower temperatufelimil, it seemthal insufficientaclivation
enerSy was prcduced that allow the critical process
(d€crcase
the effectiveness
ofCGTaseproductioD)to mcur
while,
at
the
upper
limit. the increment of the
ll0l
prodDction rate as proporl;onal Io the lemperature
increnrentwas oUset by the changesin prctein structures
238
resulting inactivation or denaturalion of critical cell
proleinsiwilh an accompmyingcessation
ofgrowth.
Tbe alkalinityof the growth environmentof the microbes
ds response!o the spec;ficchangesin proteinstructures.
ln one study,the rcsponseof an alkali-lolerant,a.ill!r sp.
!o growlh at pH 7.5 and 10.2,it was shown rhartherewere
quantitativediffercnces in the cell membmne protein
conpositionsIlll Studieson an alkalopb;lic,a.ir6 sp.
ISBN:983-2643l5-5
Pneedi^ss
fiat grow at pll 10.0 and 8.2 also show ihal there were
speclflcdifferencein rhe prcteincompolitions.which were
reflectedin the greaterlegativecbargeof the proteinsrl pH
10.0
tl2ltl3t.
Similarly,analysisof the cell envelopeof a largenumberol
alkalophilicBacill,rr sp. show ftat in some shins there
were largeamountsof negativelycharsedcompounds{141.
This may be one of the redons the cell membranercquired
sh;le
alkaline pH for the aansport charged substances.
pH
at
neulral
funclion
optimally
I15l
intracellularenzymes
Conclusion
The optimisationof CGTase p.oductionb.!-'Ba.illlr sp.
TSI-I was conductedi| batchcullure.From the Response
SurfaceMethodolosy(RSM), the optimum condirionsfor
and pH obsered to be 29.59'Cand 10.32,
boih temperature
where tfie expecrationof CGTasepoduction was 76.17
U/ml. Wbile from the experimenlapplied, the CGTase
p.oductionobtainedto be 75.48 U/ml, which was closeto
the modelpredictior.
Acknowledgement
Tbefullysuppo by Assoc.Dr. RosliMd. IuiasandDr'
Mdihah Md. Sallehwas gmlefully acknowledged.
References
tll S@rli J. 1998. lntroductionand generaloverview of
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t4l Kaneko T., Kato T., NakamuraN. and Horikoshi K.
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ISBN:983-2643-15-5
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[6] Kitahah S., Tsuyuma N. and Okada S 1973
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ll5lOhlaK., KiyomiyaA., Koyma N. andNoschY. 1975.
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239