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(CGTase)from Bacillus
Production of CyclodextrinGlucanotransferase
HRI in ShakeFlasks
steqrothermophilus
Wong Tien Eel
Madihah Md. SaUe
RoshanidaAbd Rahman2 Rosli Md' Illias3
OsmanHassats KamarulzamanKamaruildin6
Nik Azmi Nik MahmoodT
anll Natural ResourcesEnSnleentS
Depa hlent oJ BiaPncessEnSineerihS, Facub! oJ Chemical EiSineerinS
Ltnireliti TelatologiMatavsia S13I0 UTM Skudai Johor'Matalsid
Tel: 60-7 5535472Fd: 6'07'5581163
IEnaiL: tute@ ar i ng n!
Ptli
t Enait: ranida@utn m!
j
Enail: rtosli@utnmY
Univ
aDepartntentof BioloS! Facultf of
'ci%ce
ili Teknala7iMalavsia 813l0 UTM Skudai,Johar'Matavsid
Tel:60-7 55313208tu6it: ntadihah@kinioJiutn n!
5 Schaotof Bioscienceaid Biotechnologr' Facuk! oJScienceand Techaotoqv
'
I|nirersitiKeborysaanMalavsit Bargi, S.Lat8o\ Malarsia
ichemical ondIfl.lusnial BiotechhaloSvCentre, SIRIM Befi^tl, Malafsia
Introduction
Abstract
Ttu objecliv aJ this papet is to incrcase CGTase
producrion hont Bacilhts steatuthernopllilus HRI bl
optidi.ation oJ ne.tiu'n .fotnulation and fetuPnlatiah
;ariables. Ceitrct conPosirc Jactorial desisn Nas sed t'r
ochievethispurpose lnterpretationoi resuuswat 'atned
(ANOVA)and anettzed
okt usinshe anb,sis of 1)ariattce
sutace
using the 3-D rcspoase
PIot The paranete^
h
the
derliun
studied
foffiulation werc concentratton oJ
v& na,ch, pcptaneca.cin, KTHPO, CaCI, orul initiol pH
ol the neaiun. lncrcav ia lhe enqhe acttviDup to 14 2A
U/ml was obseped frcn the nodeL of erperinent&t design
conDared to the initial activitv of 420 U/ml, with the
restlexion constan (F) of 0.9186 For oplinization of PH
a;d aAitatiorspccd.cuhud qa, incubatcdin shakcla"k
ar S iC. CCr a ' e p t.4 k ? d u a s 1 6 1 5 U /n l . Li l h a
rcgression o192.34Eo. An incrcase of 3.8 fod in ccTdse
We heve
activit\) eas obtained aJkt optinizatio,l
succe;sfulty optinized the pr.'ductin ol Ccrase fton
Bocillus stearothernlophilus HRI usinS erpennentat
Keywords:
stearcthernoPhilus
CGTase. aa.illdr
Su.faceMerhodolosy.
Response
Oplimiation.
Cyclodexrrin(CD) is a closed-ringsltucturecontatningslx'
sevenor eigh! glucoseunitsand knorvnas ct-, P- andY CD'
respectivelyI1l. The inteno. of CDs is relarivelvaPolar
compared to water, and thereby CDs can easily form
which can
inclusioncomplexwnh manyorganjcsubstances
gues!
the
prcperties
of
physicochemical
change the
moleculeilhus increasingtheirwatersolubilitvand stabitity
12. 3, 4. 51. These prcPenies made CDs became
increasingly imponant fof industrial application particularly
dairy and cosmeticsindusrrv[4]
ir fmd, pharmaceuricd.
CDs are producG of starch degradalion by cvclodextrin
(CGTase; EC 24119), a tyPe of
elucanotransfemse
ixtracellular enzyme excret€d by bacleria. CGTase is
producen by vsrious bacterial speciesand mostlv reported
are fmm BacUlt species.Microorganisms such as adcill&s
stearotheruphilus 16l,Bacillus autolyticus11149[?] and
Bacillus nceftr\
tSl are known lo produce CCTase.
Production of CCTase depends much on the reaciion of
producer organisns towards substrale aDd fermenhton
The objectiveof this sludy is to optimiseproductionofthe
CCTas€ enzyme from Adcillus stearcthennophilus HRt by
using design of €xperinent method Malhematical models
were developed to conElate sll the facto6 lnalysed and to
obtain the optimal conditions for CGTase production.
.ii
222
I 5-5
ISBN:98.1-2641-
t
T
2
Conf.ttncc Or Ch.rtidl .
Aiaprc.es Eneate?tn1!
Praceetlrtss af hrtntutittol
271 2qL Aqu! 20oi uDirr:ili M.trtria sabah,K.tu KntaLrrll
Materials and Melhods
Preparationof Eacterial Inoculunt
\a.illur xeatuthemophilur HRI were gro$'n in 20 ml
mcd;um containing29. (s/v) sago starch, 1.7570(w/v)
pepbne fron meat.0.l% (w/v) K,HPOaand 0.027. (w/v)
MgSoa.TH,Owith initial pH 7.2 in a 250 ml conical flask.
The culturewas incubatedat 50'C whh shakingat 200 rpm
ior 18 houn. l'he cells rverc ihen harvesled by
c€nl.ifugalioDar 5000 rpm fo. 5 minutesand washedonce
wirh normalsalinesolutionro Eivean opticaldensity(OD)
rclding of0.5 ar 550 nnr.
experiments. The desien rlso takes iDto accounr 2
replicates,2 sor poi!'ts and 3 centrepoints. The coded
lalues for differei't variablesare shownir Table2 and e
desiSi mar.ix chosentbr rhe ioirial studyare showr in (hc
Table4. Response
sufacc plots weregenerated
by Design
LrperrrS r.' tF-.e.
LIS A V
. cr.6.0.41.ofr$J.e.
Tabt.2 - Code.lvatL.sJo{enthtatian wridbks
High
InnialpH,
A
PreparatioDof C.ude DnzJ'ne
Ten percentof aa.llllJ r/edlr/ls',ntpnillir HRI inocnlum
$ a ( J lr i\ r r ed at \ 0 o . l u r .l r h o u r. .;(h ..n ri n uor.
shaking in 500 ml conical flask containinS 100 ml of
mediun. After cultivation, the cells were removed by
centrifugationar 5000 en for 2 minutes.The supernatanl
rlus Lrsedas crude enzymesolution for assayiDgenzyme
€nzyme aciivity lvas deF.nrined using phenolphthalein
delhod b) Kanekol9l. .
ExperimentalDesignand OptimizatioD
Designofexperiment ll0l was usedto show the slatistical
signiUcanceof ihe vafiables such as conccntrationof
carbon. nitrogen, phosphoms and nriDeral salt sources
includinginilial pH in the neditlm fomulation of CCTase
production.Responsesurface methodologywrs used to
indicate the optimum condiiion of the variablesand to
understand
tfie intemclionbelweenrwo variables[l1].
The low, middle and high.leyels of each vdiable werc
given in Table l A 2'' fractional faclorial centrul
conposite design for five indeperdenlvariableswitb six
stff poinrs(a 2) and six replicationof the cenkal points.
leadingto a total32 setsofexpe.imentswerc caried out in
this study.
Tabte 1 tzvet af vatiables far nediunfomutarion
High
Sasostarch(9,{).Xl
l5
20
t0
0.6
KTHPO4
G/l), Xl
CaCl,Gn),xa
InirialpH,
x5
0.8
'1.6
o.2
lnitial scie€ningdiscoveredthat 55'C yields the highest
CGTase compnred ro othe! temperaturesll2l.
Thus,
cultures were incubaled al this lemperature for the
optimizationof fermentationvariables(pH and agitadon
speed). This processwas performed using a 23full factorial
cenr&l composiredesign, leadinS to a lotal 15 sets of
ISBN:983-2&3-15-5
10.0
5.0
2l\)
t00
From the expe.imenhl resulls, an app.oxinrate
polynonrial's reladonship lor dependent variabtes of
C C T..e acri .r) ^.., obr.' rnpd.
l he oe rgr e\pcrin( . . ,
were caried oul for anrlysis by using Design Expefi
software (S!a!e-Ease Inc.. Statistics made easy,
Minneapolis,MN, US. Ve6ion 6.0.4).
Resultsand Discussions
Assat ofCGTase
Peprcne
casein(g/l),X,
Loiv
Experimen$ with diiferent combinaton of sago sta,.h,
peptooeiro.r casein,KrHPOr, Caclr and initial pH were
performed.CGTase activity was lssayed lnd cllcula|ed
afret Ba.illus steairhenlophilw HRI inoculuD was
cultivatedat 50'C for 24 hours.This .esuh was analysed
usi nerheandl )sr.oi rJri .ncprA N O\ A , . dpp.opr dr rco
tbe expefimental design used. The coefficieil of
determination,
R' oblai|ed was 0.9486which iDplies that
fte sampleva.iationof 9.1.869d
lbr CGTaseprcductionis
attributedto the independent
variablesrested.The R'?value
also ;ndicalesthat only 5.147oof dretotal variationwas not
explainedby the model.
The regr€ssion
equation(Eq. l) obtainedafter analysisof
varianceg;ves the productionof CGTasefrcm Aa.iller
stearothemophilusHRl as a funclion of the different
variable! sago starch (Xr, s/), peptonefrom casein(X,,
s/l), K,HPoa (Xr. gl), Cacl, (Xa. 91) and initial pH (x5).
All tenns regardlessof their significancewas includedin
the followingsecondorderpolynomialeqlation:
Y = 11.84- 0.81Xr+ t.02X' +0.43Xr + 0.28X4+ 0.70>i5
l.58xr'?- 0.85xr'?- O.l3xj'z- 0.46x4'1, 1.62x51,
0.56XrX, - 0.37X1Xr + 0.17Xr& - 1.01XrX5 +
0.050X,& - 0.33X,)q + 0.40XrX5- 0.44XrXa +
(t)
0.018x3x5-0.96)i{x5
Regression
modelwith I offset.5 linear,5quadraticand 10
irteractiontermswas employedby usingthe DesignExpet
When an optinisaiionprcgrammewas run within the lested
range,the oplimum levels of the variablesobtainedwerc
16.02g/i saso starch,20 g/l peptonefrcm casein,1.4 g/l
KrHPOa,0.2g/l Caclz and pH 7.5.1.The concentration
of
nitrogensource(pharmamedia
in their study) was at 21.0
by using
sn for production of CGTasefrom da.,-llKrrurr
experimentaldesign [4]. Bes;des.Gawandeand Patkarin
thejr sludy found that the concentrationof mineral salts
223
Pt.ceal]ss
.f tncmatiatul Canfeftnte an chc'ti.al nn.l Ri.rtue.s Engin?ni1s
27r' ?t" Atsri?4oJ, Univ^ni MdtllsiaS.brh Krt) Krxrbdh
used {m|gnesium sulphatein rheir case) sas 05 e/l to
increase the CGTase prcduclion frc$ KLebsieLla
design[ 13]
AS 22 by osingexperimental
pneatnoniae
Fisure 2- EIlect oJpH an.l asitation spee.l an CCTase
p ra!1u.1i oti b! I ac illus stea rothem oPhi I us HRI
Figure1- Elfe.t af pept.,ne Uiom caseh) c.ncentrano ahd
KIHPOa conceattuiotl o, CGTasePtuduction bl Bdcillus
st4rcthemo7hihs HRI
The naximum responseptedicted from the model was
14.20 U/ml of CGTase. Repeated experiments were
performed for the produciion of CGTase by Bz.Iltur
stearathetuphilus HRI cultivated in the oplimised
mediunr.The experimentalresultsfrom three replications
gave CGTaseaclivity of 14.80 U/ml a.d fte model was
provento beadequnre.
For the fermentationvariablesoplimization.the observed
enzymeactivity was conelatedlvith experimentalvariables
CCTase =
15.25+ 0.854 - 2.618 - 5.46A'? 2.l3Br O,?4AB
Q)
r'/hereA=pH and B= erll. For thk equation,the Rz value
of 0.9234 confirmed tha! the variabilily in the enzyme
activity could be associatedto the experimenraltaciorsto
the exrentof92.347., with only 7.667,mt explainedby the
model.
The optinum levels for pH and agitadonspeedwere 7.54
and 120 em respectively.lJe and Chen discoaeredthat
Asper?ilus niser prcd\.ed CGTase in the pH range of 5.0
!o 8.0 tl4l. However,CGTaF productionis best at the
nefir^l bne fot B. stearothemophilu: HRl.
224
Experimentalresulls of three replicalionsgave CGTase
acliviry of 16.15 U/ml, which is close Io rhe prcdicted
modelvalueof 16.08U/ml. This is a 3.8 fold incrcasefrom
the initial CGTase aclivity of 4.20U/m1.Thus, CGTase
beenoplinized.
prcductionhassuccesstully
Conclusion
CGTase from Bacillus stedrothemophilur aa] had been
optim;zedwith overau3-8 fold increasein the
successtully
rcLivily. Optimization of the medium had increased
Furtbel
CCTase fmm 4.20 U/n to 14.80 U/ml.
optimization of fementation va.iables lhus increased
CGTaseto 16.15U/ml. The coelTicienlof det€rminalon
modelswereabove90% and this Proved
for bothregression
rhar rhe equar:onsgenerale,lD) e\peri ne. r.' Jes;gn wete
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