The effect of pH and temperature were investigated for an optimized condition of cyclodextrin glucanotransferase (CGTase) production from Bacillus sp. TS1-1. The optimization process was analysed using Central Composite Experimental Design in Response Surface Methodology (RSM) by Design Expert Version 6.0.4 (StatEase, USA). This design was employed to derive a statistical model for the effects of pH and temperature on the production of CGTase from Bacillus sp. TS1-1. The coefficient of determination, R2 was found to be 97.85%. The temperature of 29.59C and the pH of 10.32 had been found to be the optimum conditions for the maximum production of CGTase after 24 hr of incubation. In these conditions, the CGTase activity was attained to be 75.48 U/ml, which was close to the model predictions

Abdul Aziz, Suriani

Abdul Rahman, Roshanida

Ahmad Tarmizi, Azmil Haizam

Hassan, Osman

Kamaruddin, Kamarulzaman

Md. Illias, Rosli

Md. Salleh, Madihah

English

Universiti Teknologi Malaysia Institutional Repository

p,$e. l s, 6 tie nnli,it ucuri;;; Ii:,:,::::;i i";:tri,:H;:;;:i:l;'[i:i:iProduction of Cyclodextrin Glucanotransfcrase (CG'fase) from Bacillas sp' TS1-1:Process 0PtimisationAzmil Haizam Ahmad Tarmizir Rosli Md. Illias2 Madihah Md' Salleh3 Osman Hassana- -r.-"rrir"ti"t ramaruddinj Roshanida AbdulRahnran6 Suraini Abdul AzizTFatuLt! afChenicoL E^gittt.riry and NaItu ol Resoutces EkgineennS'Ur^,!rtiti TeknabBi Matutskt, 8ll lA Ska.ldi, Johor Maldlsi.t.TeL 6A-7 -55 3 5172. Fa\: 6'07'558 I463Enn iL :hdi za n1,79 @ la h o a. o fi'Enoil: ftasli@ktnhtY6 Ehail: ranida@ 4.n!rD.P|nnent oJ Biata$Facrlt! o[Scienc.,Ltn^ e rsiti Teknolagi Mak 'rid,3l3IA Skdai, Johat, MtktltidTe I : 60-7 - 5 5 3$2A. Entt i I : t natl ihah @ ki nia li. tu n ta'laSchaot aJ CheDtit:dL and FoodSciences,Facult! af Science atld Techtnlog',Urive$i t i Kebdnllsaan MaLorsia, 430U Bangi, SekutSoL Malavtia5Bioprccess an.I CherticaL TechMloS! CentreSIRIM Rerhad, Malalsi(tl Faculty of Food 7'cc1"1o1os! anl Food Science,Unire,si ti Putn Malaysia, 4 3100 Setuans, SelanSol MalavsiaTel: 60-3 -89468367. Far: 6'03'89423552AbstractIhe effect d pH a^LI rcnperatutu flere iirestis.lvdfor onoptnrked condition oJ cycLodextth Slucanota tJeruseGATdse) pra.lu.tion fron BdcilLus sp. TSI t Thcaptimisation process |9as anaLlsed using CenttulCanpasite ExperinlnnL Deiisn h ResPo$e Su'faceLlethodoloty (RSM) by Desi|, Eqert V.Nian 60.4(StatEase, USA). This .Iesip was enpLoled to d.nre astatistical tnodel for the eJIec^ of pH an.l tetnPerctrre anthe pto.luctian oJ Ccrase fron BaciLlus sp. TSI'1. Thecoe:licient o.f .letenintion, Rz was found to be 97 851o.n1e rcnlpenture of29.5q'C and te PH oJ 10.32 had beenlau4d Io be ttp aptidtunt.onJ'lioht lbr th. ncLtihunproduction of CGTase aJter 21 hours of iac\bation tn theseconditiohs, the CGTase activitf wat diaine.l to be 75 46Luhll. which \|6 clop ta tlz h1odel Prc.lictionsKeywords:Cyclodextrin Glucanotmnsferase, Optimisalion Condition.COTrse Acdvity, Prcduction. Response surfaceMethodologyIntroductionCyclodextrin glucanotransfeLase, CGTase (EC 2.4 t 19) hasbeen found in several bacterinl species and catalyses in bothiniermolecula. and int.amolecula. transglycosylation of o'1,,1 glucan. The reaction between CGTase wilh starchproduced cyclodextrins (CDs) with variety ofmalooligosaccharides tll. CGTase is a mulliiunctionalenzyme, whercas bes;des cyclization (!he conversion olslarch and relared o-1, 4-glucans into cDs thrcugh aninlmmolecular transglycosylatiot reaction) this enzymemana8es to catalyse a coupling rcaction (opening of CDrings and lmnsfering of linear mallooligosacchandes toacceptos) ihrough intermol@ular rransglycosylalronreactions- Nevedheless, CGT$e had a week contribuliontowards hydrclysing activity wilh water mol€cules t2l.Cyclodextr;ns (CDg have a structure of oligosacchariderings comprjs;ns '1-1. 4linked slucopymnose re$d!es.Cyclodextrins are also known as cycloamyloses orSchardinger Dext.ins. The are mainly thr@ common typesof CDs. which a'€ made up of 6, 7 or 8 molecules,identified as !-, ll- and u-cyclodextrins resp4tively CDmolecules have a torueshaped structures, wherc thehydrophilie hydroxyl groups at the exterior site and thehydrophobic C-H sroups and glycosidic oxygen d theinteriorcavity. lD the recent years. the requhements ofCDshave received grear attentions, especially in food,pha.maceutical, chemical. cosmetic as well as theasricultural induslries i3l. The reason being is dre capableofCDs molecules to fomr incllsion complexes with a widevariety of guest molecules (organic or inorganic) withou!changing the properties of fie guesl molecule ir selves.CDs are atso capable to slabilize labile materials, mask off236 ISBN:983-2643-15-5Pr.ct.tlhSs af Inxmotiona! Ca'!f.En.e Ar Ch.mi.al and BiL?taces Et$nt1titl!27r' 2qt Aut:d?003, U,tnPt\itiilolnrsia Sabrh. Kttt Kt)atalrodous, emulsity oils and chdge viscous or oilycomPounds into powde..Among the th.ee main types of CDs produced, D-CD isrnore widely used and developed tbr various applications.Owing !o ils low solubility in water, -l-CD is readilyseparated from lhe rcaction mixture withoul any lreatmentwirh orSanic solveDts and ils inclusion complexes can beeasily prcpared. The capability of predominantly !-CDproduciDg can reduce subseqtrerl purification costs, leadingto commerc;ally desirabilily. Therefore. effofts are lbcusedon finding the suitable CGTase produce$ for an efficient of'CD production.Approach arld methodsCulture ConditioN for CGTase ProductionAo.ill!.r sp. TSI I was grown in optimised mediunr withrhe composition of 2% ($lv) s.so starch. 19. (w/v) yeastextract. 0.1% (w/v) K,HPOa atd 0.02Eo {w/v) MgSOr.?HlO and added with l0% (w/v) ofNa,COr separately.depending on the pH ol fte medium. The cells werec ltLrred in 250 ml conicrl flasks and incubaled at |heEmpe.arure of 37oC wilb the agitation of 200 rym ior 18hours. The cells werc then centnf!8ed at 501ru em ibr 4 to5 minutes, walhed once with no.mal saline solution (0.85'.4Nacl), giving a lu.bidi!y (oplical density) of0.5 41600 nm.About l07o of bacterial inmulum was inoculated inlo themedjum. giv;ng rhe total volume of 100 ml (including themedium) in each 500 ml conical flasks- The cultures werethen incubated al the rotation speed of 100 rpn wilblemperaturc shown in Table I fo. 48 houn in an orbildshaker. Samples were hanesled every 2 houn for the fi6124 hours ard every 4 hours for the next 24 bou.s. ln everysamplirg, rhe sample was centrifuged at 5000 rpm for 2minutes to separate lhe bacterial cell and the clearCGTase AssayThe CGTas€ aclivity was measured using ihe methodesrablished by t4l. The .qtction nixlures containing 40 mgof soluble starch in 1.0 ml of 0.1 M sodium phospha€buffer (pH 6.0) and 0.5 ml supernatant was ancubated al60"C fbr l0 minutes. The reaction was stopped by adding3.5 ml of 30 mM NaOH, followed 0.5 ml of 0.0?% (wv)phenolphthalein in J mM Narcq solution- rhe colourinFnsity of the samples was measurcd al 550 nm aflerslanding up for 15 minutes at room temperature. The blanksolution, which lack of enzyme (m€dium) was prepared loreach batch of assays. Standard curve using 0.1% v of ll-ResultsFrom the experimental design, experiments with differentcombination of temperaturc and pH were performed. TheCGTase activities of each s€ts of experiment wered€termined after he cul$re was cultivat€d for 24 hours.The equation obtained afler analysis of variance (ANOVA)CD (Fluka) to repLace soluble strrch and c.ude enzymere$ecdvely was plotted. A unit of erzylre acllvrry wrsdefined as the amounl of enzyme thal forms I $nol of FDrt Cell Weight D€terminalionThe.e werc rwo approaches in lhe cell mrss dererminltion,depending to lhe starch presence in the culture- li theculture contains sta.ch. 0.L ml enzynre --anylase (NovoNordisk) was added 1o I ml of culturc to hydrolyse theresidue starch to soluble sugars [5]. Tfie mixlure wasincubated at 100'C fof 20 nrinutes and centrifuged at 3000efl for 30 minures. The supenritanl was thcn decanledand the cel ls sere f ihered onio prc$eighted 0.2 _imcellulose nifiaG fiher (Wh.tmrn). $ashed twice withdistilied watef lnd dried in rn oven aI 95!C ibr 24 hours.Fresh medium sas trexted similady and used as blaik forreduction ofstarch impudtics in lhe sanrple.Il srarch was depleted frorn the samplc, rhe cell mass wasnreasured by taking I ml ofcuhure and cenr.iluged ar 5000rpm for 3 minutes in a microcentrifuge. The cell pellel waswashed twice with saline solution aDd died in vacuundrier, lollowed by drying in the oven at E0 "c to constartthe cell weight- The presence of slarch in the cuhtrre can bcdelemined by using iodine solution.Starch ConcentrationStarch concentmtion in lhe sample was canied outacco.ding to lbe method of t6l. 1.0 ml of srpernatant wasmixed sirh 4 ml ol  001 M iodi le | l  025 M pords. iur ,iodide (KI) and diluted wirh 15 ml of distilled waler. Thecolonr intensity was measured al 465 nm against blank ofdistilled water treated in lhe same nanner above. The starchin culture filtrate was quantified accordinS to the slandardcurve of sttuch belween 0 to 3.5 mg/nrl.Protein ContentProtein conrent was determ;ned according to ModifiedLwD' Prcrein A,sa) I7l.Exp€rimental DerignExperimenlal design was determjned based on lheestablisbed method t8lt9l. Tbe effect of temperalure andpH were studied using Central Composite Des;en inResponse Surface Methodology (RSM). The optimisationwas performed to the total sets of l7 experiments,consistin8 four factorial poinls, eight axial (star) points andIive cente points. Tbe ranges chosen for botr processvariables were 9 to ll for pH and 25oC to 35'C forremperature, The CCTase aclivily was chosen a1 thedu.ation of 24 hour in every run of experimenls.gives the CGTase produciion from Ba.ir6 sp. TSI-1 as afunction of lemperatu.e and pH. The final equaiion (actualvalue) w6 exprcssed in second order polynomial equarion0).X= 2252.35464+51.59356A+301.62010B-0.90418A?-15.05414B1 + 0.27550A8(l) where X=CGTde activiry, U/ml, A=Tempera re, "Cand B=pH.ISBN:981-2643-15-5 237The rhree dinreisional presentation (co.relxtion berweenreDpemrure rnd pll on rhe ccTase production) lvas shownin Figure l. The optimnm temperature and pH lbr lhemaximun CGT.se production werc 29.59'C and 10.32respecrively, giving ! prcdicted CGTrse production of76.17 U/ml.The op{inru|r conditions for both rempenture rnd pH wereapplied in the sh.ke ilasks to conrpare lhe CGTasep,!duction with the predicted or€s. BeskLes, lhe grcwthkinctics of the nicrobc was also studied. The CCTaseprcducrion at 24 houlS incubation was oblained Io be 75.48Ll/,nt. whi.h $rs closed to the nrodel had p'tdicted. TheCGTase producrion iourrl ro be consrant atter l8 bou6 ofDiscussionThe CGT.se production .s a function of lemperalure andpH is $rosn in equntion (l). The fitness of the nrodel rvasexpessed by rhe coefilcienr of determination, Rr. The R2value, 0.9785 oL 97.85'Z indicate the !a.iability in thePnaalklt .l httltti.krl Ca'*ratu On ChentetL tnd Biopn..s Erlittitt27' 2q Arfui 2AA3 . Urliftrxiti M.la,"sh Sahah Kotu Ktnnnhrcsponse of the model, whe.e 2-15610 of lhe total varirtionwas not explained by tlre model (equation-l). The value ofadiusled deteflnination coefticient (Adi R' = 0.9687) wasfound to be hish lnd .dvocated for a hish sisnificance of1be model. The coirelation coefficient. R lvas mai.ly usedto show the corelation measu.ement of estimation in lheregression model. The closer the R !.lue to the unity, thebeuer co.Lelation between the obseNed and the predictedvalues was gain. From ANOVA, the R value was found !obe0.98,12.As mentjoned pfeviously, the oplimum conditions ibrmllximum CCTrse prcduction lvere pH 10.12 andlempemture of 29.6"C, giving n predicrod value ot 76.17U/ml. From lhe rwo-dimensional pr€seirarion of drercsponse sudace, it was observed rat the optlnum CGTaseproduction response was more sensitive to chrnges in onepH unit rather tban one temperature nnit (t). Thereib.e.lhe response su.frce analysis of Lhe experimentrl rcsulrscleirly reveals rhe intenelationship belveen reinperarureand pH as afecting the CGTase prodlction.39 930327 7715Figure I : The lhrce-dnnensio'1al presentatian of the rcspanse surface for th. CGTdse auiyirJ of Bacillus sp. TS I 1 . Tenperdtureunits are in.legrees Celcirswhile the CGTase activit! unns werc i1t U/nl.76 4m764 2494CGTase Act. 52.oso1The lemp€rature above o. below lhe oplimum temperature(29 6 C) was observed ro give low CGTase produclion. A1lower temperatufe limil, it seem thal insufficient aclivationenerSy was prcduced that allow the critical process(d€crcase the effectiveness ofCGTase productioD) to mcurll0l while, at the upper limit. the increment of theprodDction rate as proporl;onal Io the lemperatureincrenrent was oUset by the changes in prctein structuresresulting inactivation or denaturalion of critical cellproleinsi wilh an accompmying cessation ofgrowth.Tbe alkalinity of the growth environment of the microbesds response !o the spec;fic changes in protein structures.ln one study, the rcsponse of an alkali-lolerant ,a.ill!r sp.!o growlh at pH 7.5 and 10.2, it was shown rhar there werequantitative differcnces in the cell membmne proteinconpositions Illl Studies on an alkalopb;lic ,a.ir6 sp.238 ISBN:983-2643- l5-5fiat grow at pll 10.0 and 8.2 also show ihal there werespeclflc difference in rhe prctein compolitions. which werereflected in the greater legative cbarge of the proteins rl pH10.0 t l2lt l3t.Similarly, analysis of the cell envelope of a large number olalkalophilic Bacill,rr sp. show ftat in some shins therewere large amounts of negatively charsed compounds {141.This may be one of the redons the cell membrane rcquiredalkaline pH for the aansport charged substances. sh;leintracellular enzymes funclion optimally at neulral pH I15lConclusionThe optimisation of CGTase p.oduction b.!-' Ba.illlr sp.TSI-I was conducted i| batch cullure. From the ResponseSurface Methodolosy (RSM), the optimum condirions forboih temperature and pH obsered to be 29.59'C and 10.32,where tfie expecration of CGTase poduction was 76.17U/ml. Wbile from the experimenl applied, the CGTasep.oduction obtained to be 75.48 U/ml, which was close tothe modelpredictior.AcknowledgementTbe fully suppo by Assoc. Dr. Rosli Md. Iuias and Dr'Mdihah Md. Salleh was gmlefully acknowledged.Referencestll S@rli J. 1998. lntroduction and general overview ofcyclodextrin chemisrry Chem. Ret'. 98:17 43't'153[2] Tonkova A. 1998. Bacterial CyclodextnnGlucanotransferase. Erctme and MicrobialT e c h n o I o I r. 22 t6'7 8 -686.[3] A. Biwe., G. Antranikian. E. Heinzle 2002. Bnzymaticproduction of cyclodextrins. Appl MicrobiolB i o t e c h n o l. 59.609 -6 17.t4l Kaneko T., Kato T., Nakamura N. and Horikoshi K.1987. Spectrophotometric Determiration ofCyclization Activity of il'Cyclodextrin-Fo.mit8Pneedi^ss ol IntznntionaL canI..ohe on chatud anrl Biort.Qs En,ilt. tki2/ -2q' AuEti2AA3. Unive^iti tlok]-:id Sabah. Kala KinLb.trCyclodexrin Glucanotransfe.ase. J. Jpn.,to.S/dr.,t J.t.29:4548.t5l Anff A.8., Kadm M.1.. Madihah M.S., Sahaid K.Mand Suriani A. 2001. Direct Fermentatron olGelatinized Sago Starch 1a Acercne-Butanol_Ethanolby Clostridium acertobutylicun Wotkl JourMl ofM icnbioloey otld B iarcchnoLoqy l7.l-lo[6] Kitahah S., Tsuyuma N. and Okada S 1973Purificadon and some properties of cyclodextnnglycosyltnnsfemse from the $ain of Ba.llrlr sP.Oumal of Agricultuml Biological Chemistry.38(2):387-393.[?] Pierce Chemical Company. (1996). Modified LolvryPro.ein A\-)  .  Ro.kford.  U.S.A. In ' r ru '  .onl8l Haalaid P.D. (1989). Expenmental Design inBiolechnology . New York. Marcel Dekker.tgl Montgomery D.C. Second Edition. t992. D.sitln andAnallsis E peinents. Ne|t York. John wiley & Sonslnc. 540.li0lCaldwell D.R. 1995. Mictobiat Phlsiotas! andMerarolk,n. lA Wm C Brorvn Communications. Inc.IlllKoyama N., Takinishi H. and Nosoh Y. 1981. Apossible relation of membrane proteins of theaLkalostabililyof a facullatively alkalophilic Bd.illur.FEMS MictobioloSy Lette rs. 16:213-216.ll2lKoyana N. and Nosoh Y. 1976. The effect of the pHof culture medium on lhe alkalopbilicity ofa species ofBacillus. Achieres of Micrcbtolos). 109: 105' 108.tl3lKoyama N, Kiyomiya A. snd Nosoh Y. 19?6. Na1dependant uplake of amino acids by an alkalophilicBacillus. FEBS LztIe$. 72:7 7 -l LIt4lHorikoshi K. and Akiba T. 1982. AlkalophiticMicrco.ganisns- Spnnger. Berlin. Heidelb€.g andll5lOhlaK., KiyomiyaA., Koyma N. and Nosch Y. 1975.The basis of the alkalophilic property of a species ofBacillus. Joumal of Genetut Microbiolosy. 86.259266.ISBN:983-2643-15-5 239

Production of cyclodextrin glucanotransferase (CGTase) from Bacillus sp. TS1-1: process optimisation

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Production of cyclodextrin glucanotransferase (CGTase) from Bacillus sp. TS1-1: process optimisation

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