p,F?r,c. ath.n:5o41 ar! i :,i:::,; ; t:",:;:::;:, ; :;"i::;;:i "c ;:;;:" (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 References S.,Dijkuizen,L., Dijkstra,B.. Jensen.B and I1l Pedersen, Jurgensen,' S. 1995. A Bett€r Bnzyme lor Cyclodexr.ins. Clefrrern. Dec: 19-25. [2] Kim, Tae-Jong,Ire, Yun'Dons and Kim, Hak-sung 1992. Productionof Cyclodextins from Unliquefied Cornstarch Using Cyclodextnn Glycosyltransferasein a Membrano Bioreactor. 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Cylcodextdn Chrcanotmnsferasettom BacillL6 stcanrhamtophihs: Purificationby Affinny Ch'omarogirplrylnd Prcpefties.K.)reh J()undL 'Is Applie.lMi.tubiol%rRiate.htrr1aS1 i8 (6): 5E5 590. t7l ToNrr. K.. Krned.. M., Karvamur^.K. and Nakafishi K ls t l P . . t . . J.o n J 1 .rP o p -| l r,, o l I C /c . o e \r , Glucanolrafsferasefrom Ba.illlrs autalvi.rs 11149 and selectile Fomation of jl-Cyclodext r. Jorlral o/ FernEhtatiDh dntl Bioenginee ritry.2. 89'92Nogrady. N.. Pocsi, l. lnd Szentirmal. A 1995. t8l Cyclodexl.in Glycosylransfefasemay be the Only Starch Degrading Enzyme in BaciIus rlaceruns. Bi t)k. hnob BI Appli ed Bi ocheni t I n 2l : 233-243. tel Kaneko,T.. Karo.T.. Nakamun.N. and Ho.ikoshi.K. 1987.Specrrophorometric dererminarion of cyclizatior acliviry of 0-cyclodextrinforming clclomaltodextrin glucanotranslerlse.J.,un1al Japan Societ! Starch Scancc.34(I ): .15-48. ISBN:983-2643-15 5 l l 0l Mo gomcfy,D . C . 1976. D csi sn and A nrtvsisof L\pcr,,e.,. 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Optinrizarionof Medium Composition for rhe P.oduction of Glucosyltnnsferase by AsperyiLlls niget wtrb Response Su.frce MerhodoloSy. En tne antl M icrobi al TechaaLoSy.2 l t 436-410. )25