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Biotechnol. Bioprocess Eng. 2000, 5: 174-180

Immobilization of Cyclodextrin Glucanotransferase on AmberliteIRA-900 for Biosynthesis of Transglycosylated Xylitol

Pan-Soo Kim1

, Hyun-Dong Shin1

, Joong-Kon Park2

, and Yong-Hyun Lee1

*1 DepartmentofGeneticEngineering,CollegeofNaturalSciences,Kyungpook NationalUniversity,Taegu702-701,Korea

2 DepartmentofChemicalEngineering,CollegeofEngineering,KyungpookNationalUniversity,Taegu702-701,Korea

Abstract Cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. was ad-sorbed on the ion exchange resin Amberlite IRA-900. The optimum conditionsfortheim-mobilization of the CGTase were pH 6.0 and 600 U CGTase/g resin, and the maximumyield of immobilization wasaround63% on the basis of the amount ratio of the adsorbedenzyme to the initial amount in the solution.Immobilization of CGTase shifted the opti-mum temperature for the enzyme to produce transglycosylated xylitol from70oCto90oC

and improved the thermalstability of immobilized CGTase,especiallyafterthe additionofsolublestarchandcalciumions.Transglycosylatedxylitol was continuously produced usingimmobilized CGTase inthecolumn type packed bed reactor,andthe operating conditionsfor maximum yield were 10%(w/v)dextrin(13 ofthe dextrose equivalent)astheglycosyldonor,10%(w/v)xylitolastheglycosylacceptor, 20 mL/h of medium flow rate,and60 oC.Themaximumyieldoftransglycosylated xylitolandproductivitywere25%and7.82gL-1h-1,respectively.The half-life of the immobilized CGTase in a column type packed bed reactorwas longer than 30 days.

Keywords: transglycosylated xylitol, transglycosylation, cyclodextrin glucanotransferase,immobilization,AmberliteIRA-900,Thermoanaerobactersp.

INTRODUCTION

Xylitol is a natural sugar alcohol composed of fivecarbons,and sweet as sugarwithnoafter-taste.Itex-hibitshigherheatstabilityandendothermicheatdur-ingsolubilizationduetoitsstructural lack of afreecar-bonylgroupcomparedtoothermono-ordi-saccharides.Itisalsomoreresistanttomicrobialdegradationandisextremely hydroscopic. It has 40% less calories thansugar, and thus can be safelyusedfordiabetics[1]. Themostbeneficialaspectofxylitolutilizationcanbefoundin its effect on dentalhealth because plaque bacteria,Streptococcusmutants,cannotutilizexylitol.Streptococcuspneumoniae, the bacterium causing ear and respiratoryinfections,is also unable toutilizexylitol.Accordingly,

xylitol iscurrentlyusedinchewinggum, confectionery,pharmaceuticals, oral hygiene products, and diabeticfoods[2].

Cyclodextrin glucanotransferase (CGTase) is an en-zyme that catalyzes the transglycosylation reaction,which transfers various glycosyl units into glycosylacceptors, such as sugars, glucose, sucrose [3], xylose,

* Corresponding authorTel: +82-53-950-5384 Fax: +82-53-959-8314e-mail:[email protected]

sorbose[4],stevioside,ascorbicacid[5],andhesperidin

[6].Theseglycosylproductsareattractiveintwoview-points; one is related to the improvement of physio-chemicalproperties and theother is thepossibilityofobtaining functional oligosaccharides [7]. CGTase hasalsobeen used forthe transglycosylation of sugar alco-hols such as the oligoglucosyl-inositol from myo-inositol[8], whichexhibits agrowthstimulatingeffectonBifidobacterium showing physiological functionality[9]. In the previous studies by the current authors[10,11],twoglycosyl-sugaralcohols, i.e., glycosyl-malti-tol and transglycosylated xylitol, were synthesizedwith the aid of CGTase. Thestructural characteristicsof the transglycosylated xylitols were analyzed byNMRand these bioproductsshowedagrowthstimula-

tion effect onBifidobacterium breve, thereby showingtheirpotentialto be alternativesweeteners.

CGTase has been immobilized by the adsorptionmethod; the adsorption of the CGTase from alkalo-philicBacillussp.onDiaionHP-20[12]anditscharac-terization in a column type bioreactor were investi-gatedforthe optimizationoftheoperationconditionsfor cyclodextrin (CD) production [13]. The CGTasefromBacillus macerans was adsorbed on IRA-93 andimmobilized in a polysulfone capillary membrane forCDproduction [14,15].Inthe previousstudies by thecurrent authors, the CGTase fromBacillus macerans


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Biotechnol. Bioprocess Eng. 2000, Vol. 5, No. 3 175

was adsorbedonAmberliteIRA-900,andtheperform-anceofthecolumn typebioreactor containing immobi-lized enzymes was evaluated with respect to the pro-duction cost of CD[16,17]. Theutilizationofimmobi-lized CGTase for a coupling reaction to transfer theglycosyl units of the donor molecules to the acceptermolecules by inter-transglycosylation has been rarely

examined although many literatures report the produc-tion of CD by the immobilized CGTase. Only onestudy reports the CGTase fromBacillus macerans ad-sorbedonDiaionTMHPA75usedfor theproductionoftransglycosylatedstevioside[18].

Inthiswork,theCGTasefromThermoanaerobactersp.was adsorbedonAmberliteIRA-900fortheproductionof transglycosylated xylitol and the optimum condi-tionsof theCGTaseimmobilization, such as thepHofthe solutionandthe mixing ratio of the enzyme to the

Amberliteresin,weredetermined.Stability and activityoftheimmobilizedenzymewerealsoinvestigated.Theeffect of the variation of operating conditions of thecolumn type packed bed reactor containing immobi-lized CGTase on the yield of transglycosylated xylitolwas investigated along with the stability of the immo-bilized enzyme in a column type packed bed reactor.This research will not only facilitate in the develop-ment of an effectivemethodfor theimmobilizationofCGTase used for an inter-transglycosylation reactionbut also improve theprocess for theefficientproduc-tionoftransglycosylatedxylitol.

MATERIALS AND METHODS

Enzyme and Substrates

ThecrudeCGTasefromThermoanaerobacter sp. (102units/mgofprotein,Novo-NordiskCo.,Denmark)wasfurther purified using an ultrafiltration kit (MWCO50,000,Amicon Co., USA) to remove impurities. Thepurified CGTase showed a specific activity of 132units/mg of protein. Xylitol (Sigma Chemical Co.,USA) was used as the glycosyl acceptor and solublestarch (Sigma Chemical Co., USA) and dextrins (Al-drichCo.,USAandSindongbangCo.,Korea)wereusedastheglycosyldonor.

Measurements of CGTase Activities

ThecouplingactivityofCGTasewasdeterminedbya modifiedYamamotosmethod[19].onemilliliterof5mM -CD,25mMsucroseand0.1mLof10mMTris-Malate-NaOHbuffer (pH6.0) solution containing thepurified CGTasewere mixed and incubatedat50oCfor30min.TheCGTase was removedbycentrifugalultra-filtration, then the reactionmixturewasmixedwith5unitsofamyloglucosidase(SigmaChemicalCo.,USA)andfurtherincubatedfor30minat50 oC.Theamountofglucose formedwasmeasuredusingaglucoseassaykit (Dongbang Co., Korea), and one unit of activitywasdefinedastheamountofenzymeproducing1mM

ofglucosepermin.Soluble starch was used as the substrate to measure

thecyclizationactivityof CGTase. one milliliterof10mM Tris-Malate-NaOH buffer (pH 6.0) solution con-taining 10%(w/v) soluble starch was mixed with 0.1mLofthepurifiedCGTase solution, then incubatedat50oC for 30 min. The produced CDs were analyzed

with HPLC,andoneunitofactivitywasdefinedastheamountofenzymeproducing1MofCDspermin.

The hydrolysis activity of CGTase was determinedby themethodofKitahata[20]. 10LofthepurifiedCGTase solution was mixed with starch (1.0% w/v)dissolved in 1.0 mL of 20mMTris-Malate-NaOH buffer(pH6.0),and incubatedfor30minat37oC.OneunitofactivitywasdefinedastheamountofCGTase thatincreased1.0%intransmittanceat660nmpermin.

Immobilization of CGTase

AmberliteIRA-900, the strong basic anion exchangeron polystyrene, waswashedwith0.5 NHClsolutionand0.5 NNaOH alkaline solution to activate theresinitself,thensuspendedin100mLof 10mMTris-Malate-NaOH buffer (pH 6.0) for equilibration. In order toimmobilize enzyme on the resin, 10gof theactivated

AmberliteIRA-900wasmixedwith6,000unitsof theCGTase dissolved in 100 mL of 20 mM Tris-Malate-NaOHbuffer(pH6.0) and incubatedat 30oC and150rpm for2h.TheoptimumpHfor theCGTaseimmobi-lization was investigated using the following buffersolutions:10 mM Glycine-HCl (pH 3.0),10 mM Cit-rate(pH4.0-5.0),10mMTris-Malate-NaOH(pH6.0),10mMKH2PO4-K2HPO4(pH7.0-8.0),10mMTris-HCl(pH 9.0), and 10 mM Glycine-NaOH (pH 10.0). Themixing ratioof theCGTase to the resin in the buffersolution variedfrom200 to 1,200unitsofCGTasepergofresin.

The pHstabilitywas investigatedbymeasuringtheresidualcouplingactivitiesofthe immobilizedand thefreeCGTasesinvariousbuffersolutionsat50oCfor1h.To measure the thermal stability of an enzyme, theresidual activity wasmeasured justafter a 30-minpre-incubation at temperature ranging from 50 to 100oC.

Additives of10mMcalciumionsand0.1%(w/v)solu-blestarchwere used in the expectation of an increase in

the thermal stability.Operation of Column Type Packed Bed Reactor

Thecolumn type packed bedreactorwascomposedof awater-jacketedglasscolumn (2.2 cm ID, 33.0cmheight),flow-ratecontroller,andwaterbath.Theglasscolumn was packed with 80 g ofAmberlite IRA-900adsorbing354 unitsofCGTasepergofresin itself. Justafter packing resins in the glass column,10mMTris-Malate-NaOHbuffer(pH6.0)was made to passthroughthe column to eliminate any unadsorbed CGTases. A


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176 Biotechnol. Bioprocess Eng. 2000, Vol. 5, No. 3

substrate solution containing 10% (w/v) dextrin and10% (w/v)xylitol dissolvedin abufferpassedthroughthe column type packed bed reactor at a flow rate rang-ing from10-50mL/h,andthetransglycosylatedxylitolproduced in the column at 60oC was detected by HPLC.

Analytical Methods

Xylitol and transglycosylated xylitol were analyzedby HPLC system (Gilson Medical Electronics, Inc.,France):Cosmosil-packedcolumn5NH2(NacalaiTesque,Inc., Japan), acetonitrile/water (65/35), 1.0 mL/min,and RI detector. The protein concentration was ana-lyzed by the Bradford method [21].

RESULTS AND DISCUSSION

Immobilization of CGTase on Amberlite IRA-900

TheCGTase purifiedbyultrafiltrationwas adsorbedonAmberliteIRA-900 resins.The well adjustedpHofthe solution during immobilization may improve the

yield of immobilization and also the stability of immo-bilized CGTase. The effect of the pH control on theimmobilization of the free CGTase andthe activityoftheadsorbedCGTasewasexamined by varyingthepHof the solutionfrom3.0to10.0 and isillustrated as thedata inFig.1(a).TheamountoftheadsorbedCGTaseincreased proportionally up to pH 6.0, due to the in-crement the negative charge of CGTase according tothe pH value, and thereafter, the adsorbed proteinreached plateauat4.2mg ofproteinpergramofresin.The activity of the adsorbed CGTase also increasedproportionally up to pH 6.0 with the increase in theadsorbedCGTase;however,itdecreased rapidlyatpHshigherthanthislevel,duemay be tothedenaturationof theCGTase at the high pH because the optimal pHof enzymeactivityis5.8 [22].TheoptimumpHfor theimmobilization of the CGTase without denaturationwas about6.0.

The mixing ratio of CGTase toAmberlite IRA-900was also found to affect the yield of immobilization and Fig. 1(b) illustrates how the mixing ratio of theCGTase to theAmberliteIRA-900 affectsontheactiv-ityof the adsorbed CGTase and the adsorption yield.10 g of Amberlite IRA-900 resin was mixed with vari-ous units of CGTase ranged from 2,000 to 12,000.The

activityoftheadsorbedCGTase increasedproportion-ally withtheamountoftheloadedCGTaseup to600units per gram of resin, and thereafter remained at aconstantlevel. From the yield data shown in Fig. 1(b),it can be easily found that the amount of adsorbedCGTase was constant and the resin was saturated withadsorbed enzyme at a mixing ratio higher than 600units per gram of resin. The saturation of the resin withenzymes resulted in the constant level of enzyme activ-ity. It can be easily concluded from the linear rela-tionship between the activity of the immobilized en-zyme and the enzyme loading at the mixing ratio lower

Fig. 1. EffectofpH(a)andthe mixingratioofCGTaseandAmberlite IRA-900, (b) on immobilization of thermostableCGTase onAmberlite IRA-900. (a) 6,000 units of CGTasewereincubatedwith10gofAmberliteIRA-900,equilibratedwithvariousbuffersof differentpHs,at30oCfor2h,andtheamountsofproteinandCGTaseactivitiesintheresins weremeasured. (b)variousamountsofCGTaserangingfrom200to1,200units ofCGTasepergofresinwereimmobilizedon10 g ofAmberlite IRA-900, equilibrated with 10 mM Tris-

Malate-NaOHbuffer (pH6.0),at30o

C for2h,andtheen-zymeactivitiesoftheresinswere measured.(!) amountofadsorbedprotein; (")activityof adsorbedCGTase; (#)ad-sorption yield.

than 300 units per gram of resin that there was no sig-nificant mass transfer resistance of substrate on theresin surface.Theoptimum mixingratioofCGTase to

Amberlite IRA-900 for the maximum activity ofimmobilized CGTase was determined as 600 units ofCGTase per gram of resin and the correspondingadsorptionyield was63%.

Enzymatic Properties of Immobilized CGTase

Theactivitiesandstabilitiesof thefreeandimmobi-lizedCGTaseatdifferent pHs ranged from3.0 to9.0were measured andshowninFig.2. Wefoundthattheoptimum pH and pH stabilitydid not change signifi-cantlyevenafter immobilization.Theeffectof there-action temperature on the three kinds of enzyme ac-tivitiesoftheimmobilizedCGTasewasalsoexamined.

As shown in Fig. 3, the activities of immobi lizedCGTase for three kinds of reactions, i.e., coupling, cycli-zation, and hydrolysis, varied according to temperaturesranged from 50oC to 100oC.Theoptimumtemperaturefor the coupling reaction shifted from 70oC to 90oC

after immobilization,however,thosefor thehydrolysisandcyclizationreactionsincreasedfrom80oCto90oC.The increase in the optimum temperature may becaused by the fact that there is a temperature gradientin the resin and the temperature inside the resin is cor-respondingly lower than the bulk temperature. How-ever, the optimum temperature for the coupling reac-tion was shifted remarkable and this may be caused byan improvement in the thermal stability and the en-forcement of the coupling activity of CGTase due tothe conformationalchangethrough the immobilizationof CGTase on the strong anion exchange resin. The


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Fig. 2. Effect of pH on activity (a) and pH stability (b) ofsolubleandimmobilizedCGTase.(a) Thecouplingactivitiesof the soluble and immobilized CGTase were measured atvariouspHs.(b) Theresidualcouplingactivitiesofthesolu-bleandimmobilizedCGTaseweremeasuredafterincubationin various pHs at 50oC for 1 h. ($) soluble CGTase; (")immobilizedCGTase.

Fig. 3. Effect of temperature on coupling (a), cyclization (b),and hydrolysis (c) activities of the soluble and immobilizedCGTase.Thecoupling,cyclizationandhydrolysisreactionsweremeasured at different temperatures from 50oC to 100oC. ($)solubleCGTase;(")immobilizedCGTase.

CGTasefromBacillusmaceranswas previouslyimmobi-lized by a similar method and used to produce CD;however, its optimum temperature decreased from55oC to 50oC after immobilization [15], showing thestrong dependence ontheoriginof theCGTase.

Thermal stabilities of the free and immobilizedCGTasetreatedatdifferenttemperatures rangedfrom50oCto100oCwerecompared and are presented inFig.4. An improvement of the thermal stability of the

CGTase afterimmobilization at the temperature above70oC is of interest. For example, the immobilizedCGTase retainedmore than80%of its initialactivityat 80oC. However, the soluble CGTase retained only55%of itsinitialactivity.Theincreasedthermalstabil-ity after immobilization seems to add to the advan-tages of the column type packed bed reactor used toproduce transglycosylated xylitol, such as improvedproductivity and the increasedreactionrate caused bythe high dilution rate of the medium in the reactor.

Two studies report that the addition of thecalciumionsand soluble starch showed a stabilizing effecton

Fig. 4. Comparisonofthermostabilityofsolubleandimmo-bilizedCGTase.Theresidualcouplingactivitiesof thesolubleandimmobilizedCGTaseweremeasuredafterpreincubating

at different temperatures for 30 min. ($) soluble CGTase;(")immobilizedCGTase.

Fig. 5. EffectofsolublestarchandCa2+ionsonthermostabil-ity of immobilized CGTase. 10 mM Ca2+and 0.1%(w/v) ofsoluble starch were added into the suspended solution con-taining 100 units of immobilized CGTase, respectively, andthe residual coupling activities were measured after pre-incubationatdifferenttemperaturesfor30min.(")control;(!)0.1%(w/v)ofsolublestarch;()10mMCa2+.

theconformation of the CGTase [23,24].10 mM cal-ciumionsand0.1%(w/v)solublestarchwereaddedtothe suspended solution of the immobilized CGTase,respectively, in the expectation that the thermal stabil-ity of the immobilized CGTase will be increased.Asshown in Fig. 5, the thermal stabilityof the immobi-lizedCGTase increasedsignificantly. For example, theresidual coupling activity increased from 60% to 85%by the addition of calcium ions andfrom60%to80%withsolublestarch at the temperature of 90oC.


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Table 1. Effectofdextroseequivalentof glycosyldonorontransglycosylationyieldandproductivityofimmobilizedCGTase

Substratesolutionof10%(w/v)donorandxylitoldissolvedin 10

mM Tris-Malate-NaOHbuffersolution(pH6.0),respectively,was

appliedtothe column type packed bed reactor(2.2 33 cm)con-taining immobilizedCGTase(354units/gresin)ataflow rate of

20mL/hand60oC.a D.E.valuesofDextrins(AldrichCo.,USA andSindongbangCo.,

Korea)weredeterminedbyLeesmethod[16].

Production of Transglycosylated Xylitol in aColumn Type Packed Bed Reactor ContainingImmobilized CGTase

We investigated how the variations of the dextroseequivalent (D.E.) value of dextrin used as the glycosyldonor, substrate concentration, and the flow rate in thecolumn type packed bed reactor would affect the trans-glycosylation yield. The degree of polymerization (D.P.)of the glycosyl donor such as starch, dextrin, andmaltooligosaccharides, is already recognized as an im-portant factor affecting the efficiency of thetransglycosylation reaction catalyzed by CGTase

[7,10,25]. Table 1 shows the effect of the D.E. value,indirectly representing the D.P. of the glycosyl donor,on theefficiency of the transglycosylation reaction inthe column type packed bed reactor containingimmobilized CGTase. The transglycosylation yieldsand productivities were maintained as about 23-24%and 7.4-7.6 gL-1h-1, respectively, at a wide range of D.E.values from 6 to 13. However, they decreasedsignificantly to 18.6% and 5.8 gL-1h-1, respectively at aD.E. value higher than 19. TheoptimumD.E. levelofdextrin wasdeterminedas13 inthis reaction system,which exhibited themaximumproductivityof7.6gL-1h-1.Theseresultsindicatethat theD.E.valueofdextrinisalso an important factor for thetransglycosylation re-

action catalyzed by immobilized CGTase, due to thedonor specificityoftheenzyme. Another study also reportsthedonor specificityofCGTase in that simple or shortchainsugars, suchasglucose,maltose,andmaltotriose,are unsuitableas theglycosyldonorin thetransglyco-sylationreaction catalyzed by theCGTase fromThermo-anaerobacter sp. [10]. However, glucose and maltosewere considered to be betterglycosylacceptors ratherthan glycosyldonorsin thetransglycosylationreactionof theCGTasefromBacillussp.[7, 25].

Fig.6showstheeffectof variation of thesubstrateconcentration from 1.0 to 15%(w/v) on the transgly-

Fig. 6. Effect of substrate concentration on transglycosyla-tionyield and productivity of the column type packed bedreactorcontaining immobilizedCGTase.Substrate solutions

composed of thesameconcentrationsofxylitolanddextrin(D.E.13) ranging from 1.0-15%(w/v) in 10 mM Tris-Malate-NaOHbuffer (pH6.0)werepassed through the col-umn type packed bed reactor(2.2 33 cm)at20mL/hand60oC. ($) transglycosylationyield;(")productivity.

cosylationyield and productivity of the column typepacked bed reactor containing immobilized CGTase.Thetransglycosylationyielddecreased as thesubstrateconcentration increased, buttheproductivityincreasedas the concentration of substrate increased up to 10%(w/v) and reached 7.8gL-1h-1.The yield of transglyco-sylation byafreeenzyme[26]and that of the CD pro-

duction by immobilized CGTase contained in thecol-umn type packed bed reactor [17] were also found to bedependent according to the substrate concentration.This phenomena is trivial because the reaction mode inthe packed bed reactor is similar to that in the batchreactor and the reaction was progressed by the first or-der reaction at low substrate concentration and by thezero order reaction at high concentration if the trans-glycosylation reaction was catalyzed by CGTase follow-ing the Michaelis-Menten kinetic mode.

Fig.7showstheeffectof thesubstrateflowrate (dilu-tion rate)ontheproductivityofthe column type packedbed reactorand thetransglycosylationyield.The trans-glycosylationyielddecreased slightlydowntoaflowrate

of20mL/hand thereafter, decreasedrapidly.In contrast,theproductivityincreased with a flow rateupto20mL/h,and thereafter, decreased gradually.As the flow rate ofthesubstrate increased,theretention time of the substratein the reactor was shortened and the transglycosylation

yield decreasedcorrespondingly. However, the productiv-ity increased and reached the maximum valueof7.8gL-1h-1 as the substrate flow rate increased up to 20mL/hbecause the mass transfer rate through the boundary layerdecreased with the flow rate. It seemed that the control-ling step is the reaction by the immobilized enzyme on thesurface of the resin at the flow rate lower than 20 mL/h

D.E. value of donorTransglycosylation

yield (%)Productivity

(gL-1h-1)

-Cyclodextrin

Soluble starchDextrin (D.E.a6)Dextrin (D.E.11)Dextrin (D.E.13)Dextrin (D.E.19)

31.1

22.223.823.724.218.6

9.72

6.947.447.407.565.81


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Fig. 7. Effect of flow rate on transglycosylationyield andproductivityofthe column type packed bed reactorcontain-ingimmobilizedCGTase.10%(w/v)substratesolutionsin 10mMTris-Malate-NaOHbuffer(pH6.0)werepassedthrough

the column type packed bed reactor(2.2 33 cm)atdifferentflowratesfrom5mL/hto50mL/hat60oC.($)transglyco-sylationyield;(")productivity.

and a conformational problem of the enzyme due to theshear stress becomes serious at a flow rate faster than 20mL/h. Accordingly, the optimum flow rate was deter-mined as 20 mL/h and was then applied for a long timeoperation for the production of transglycosylated xylitol.

Operational Stability of Immobilized CGTasePacked in Column Bioreactor

TheoperationalstabilityoftheimmobilizedCGTaseused for the production of transglycosylated xylitol inthe column type packed bed reactor during a 40-dayscontinuousoperation is illustrated in Fig. 8.Thehalf-life of theimmobilizedCGTase used for thisoperationwas 30 days, which was a little extended comparedwith 21 days of immobilized CGTase fromBacillusmacerans [14],23days ofBacillusmaceransCGTaseim-mobilized onAmberlite IRA-900 [16], and 25 days ofalkalophilicBacillussp.CGTaseimmobilizedonDiaionHP-20 [13]. Thehighoperational stabilityofthis reac-tionsystemis one of theadvantagesin theoverproduc-tionoftransglycosylatedxylitol.

CONCLUSION

The Thermoanaerobacter sp. CGTase immobilized onAmberliteIRA-900showedaresistanceagainstthermaldenaturationanda shiftto a higheroptimumtempera-ture.Moreover,the immobilizedCGTase ina columntype packed bed reactordemonstrated ahigheropera-tional stability compared to other CGTases from dif-ferent species immobilized on different materials. Withthese improved merits, the method we used in the

Fig. 8. OperationalstabilityofimmobilizedCGTaseonAm-berliteIRA-900in the column type packed bed reactor.Sub-stratesolutioncomposed of 10%(w/v)dextrin(DE13)andxylitol in 10 mM Tris-Malate-NaOH buffer (pH 6.0) was

appliedto the column type packed bed reactor (2.2 33 cm)at20mL/hand60oCfor40days.

current study appears to be effective in the overpro-ductionoftransglycosylatedxylitol.Furtherstudiesonthe kineticsof acolumn type packed bed reactor con-taining immobilized CGTase and an efficient separa-tion method to purifythe bioproduct are required forthe mass production of transglycosylated xylitol.

Acknowledgements The authors wish to acknowl-edge the financial support from the Korea ResearchFoun-dationintheprogramyearof1998.

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[Received May 1, 2000; accepted June 8, 2000]

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