Adsorption–desorption of trehalose analogues from a bioconversion mixture using activated carbon

1、Adsorptiondesorption of trehalose analogues from a bioconversion mixture using activated carbonAdsorptiondesorption of trehalose analogues from a bioconversion mixture using activated carbonChao Chen a ,Tom Desmet a ,Jef Van der Borght a ,Sze Ki Carol Lin b ,Wim Soetaert aa Center for Industrial Bio

2、technology and Biocatalysis,Department of Biochemical and Microbial Technology,Ghent University,Coupure Links 653,BE-9000Ghent,Belgium bSchool of Energy and Environment,City University of Hong Kong,Hong Konga r t i c l e i n f o Article history:Received 4April 2012Received in revised form 28May 2012

3、Accepted 29May 2012Available online 8June 2012Keywords:Trehalose analogues Activated carbon Adsorption Desorptiona b s t r a c tTrehalose (a -D -glucopyranosyl a -D -glucopyranoside)is widely used in the food industry,because of its protective effect against freezing and dehydration.Analogues of tre

4、halose have the additional benet that they are not digested and thus do not contribute to our caloric intake.Such trehalose analogues can be produced with the enzyme trehalose phosphorylase,when it is applied in the reverse,synthetic mode.A cost-effective purication procedure is,however,still lackin

5、g.Therefore,the adsorption of trehalose on activated carbon has been studied and compared with that of galactitol,i.e .the major contaminant in the process.The adsorption capacity of trehalose was found to be signicantly higher than that of galactitol,which suggested that trehalose analogs could be

6、removed from the bioconversion mixture.Selective desorption with aqueous ethanol allowed to recover the product with a purity of more than 97%.Preceding the adsorption/desorption procedure by an ion-exchange step increased the yield from 24%to 31%,but also increased the price.Therefore,the direct us

7、e of activated carbon is proposed as new strategy for the purication of enzymatically produced trehalose analogues.2012Elsevier B.V.All rights reserved.1.IntroductionTrehalose (a -D -glucopyranosyl a -D -glucopyranoside)is a non-reducing disaccharide present in a wide range of organisms (most notabl

8、y yeasts and plants),which it protects against environmental stresses such as heat,freezing and drought 1.It is also stable at a wide range of pH-values,has a mild sweet taste and is not cario-genic.These properties make it ideally suited for use in processed food preparations 2.Its synthetic analog

9、ue lactotrehalose (a -D -glucopyranosyl a -D -galactopyranoside)has as additional benet that it is not hydrolyzed by intestinal trehalase and,therefore,does not contribute to our caloric intake 3.Furthermore,lactotrehalose is a potential prebiotic because of the presence of a galactose res-idue,whic

10、h is known to stimulate the growth of bidobacteria in the human intestine 4.The biocatalytic production of lactotrehalose has been de-scribed with the use of the trehalose phosphorylase (EC 4)from Thermoanaerobacter brockii 5.The authors proposed a cou-pled reaction that starts from the cheap

11、 substrates trehalose and galactose instead of the expensive intermediate b -D -glucose-1-phosphate (b Glc1P,Fig.1).For the downstream processing,a com-bination of (bio)chemical treatments and chromatographic steps was applied.However,chromatography brings about a rather highoperational cost,prohibi

12、ting the large-scale use of trehalose ana-logues.Other purication procedures thus still need to be evaluated.Inorganic adsorption is often used to remove colors,odors and pollutants from bioprocesses 610.In terms of economic and sus-tainable criteria,granular or powdered activated carbon has been fo

13、und to be one of the best adsorbents for the majority of applica-tions 8,1117.Recently,activated carbon has been used to selectively purify maltopentaose from an aqueous solution contain-ing other maltooligosaccharides,such as maltose,maltotriose and maltotetraose 18.Because the adsorption capacity

14、of maltopentaose is lower than that of the other maltooligosaccha-rides,its purity could be increased to 75%after several adsorption cycles 19.However,adsorption alone is often not enough to obtain highly pure oligosaccharides and needs to be combined with desorption,using different ratios of water/

15、ethanol as eluent.For example,mono-and disaccharides could rst be recovered with an ethanol concentration of 515%,followed by oligosaccharides with a higher degree of polymerization (DP)at 3050%20.In this study,the purication of trehalose analogues from a bio-conversion mixture with the use of activ

16、ated carbon has been eval-uated.To that end,the sorption characteristics of trehalose as model compound was rst examined and compared with those of galactitol,i.e .the major contaminant in the process.An adsorp-tiondesorption protocol was then developed for the efcient puri-cation of lactotrehalose

17、from an enzymatic reaction.1383-5866/$-see front matter 2012Elsevier B.V.All rights reserved.Corresponding author.Tel.:+3292649921.E-mail address:chao.chenugent.be (C.Chen).2.Experimental2.1.Materials and chemicalsThe acid-washed agglomerated coal-based granular activated carbon CPG-LF 1240(see Tabl

18、e 1for properties)was a kind gift from Chemviron Carbon (USA).It was pretreated with hot deion-ized water (6070C)for 2h,dried overnight at 70C,and then kept in a desiccator.Trehalose,galactitol and other chemicals were obtained from SigmaAldrich (St.Louis,MO,USA).2.2.Equilibrium experimentsIn a 250-

19、ml glass ask,100ml of either trehalose (40mM)or galactitol (25mM)was mixed with 140%(m /v )of pretreated acti-vated carbon.The asks were shaken for 5h at 200rpm and 303K in an incubator (Multitron,Switzerland).Samples were ltered through 0.22l m nylon syringe lters before they were analyzed by HPLC.

20、The amount of the compound adsorbed by CPG-LF was calculated by Eq.(1),where q e is the amount of adsorbate per gram of adsorbent (mmol/g),V is the volume of the solution (l),M is the weight of adsorbent (g),and C 0and C e are the initial and equilib-rium concentrations in solution (mmol/l),respecti

21、vely.q e V C 0C e 12.3.Kinetic experimentsIn a 250-ml glass ask,10g of pretreated activated carbon was mixed with 50ml of deionized water.The suspension was heated to 30C and stirred at 200rpm on a magnetic hotplate stirrer (VMS-C7,VWR).Subsequently,50ml of preheated adsorbate solu-tion was added to

22、 obtain the desired initial concentration.Sampleswere collected at different times and analyzed by HPLC.The overall reduction in solution volume due to sampling was less than 2.5%,meaning that sorption rates were measured in a near-constant li-quid volume.The amount of adsorption q t (mmol/g)was cal

23、culated by Eq.(2),where C t is the liquid phase concentration at time t .q t V C 0C t 22.4.Production of lactotrehaloseLactotrehalose was produced with the trehalose phosphorylase from Caldanaerobacter subterraneus 21,using as substrates 500mM trehalose and galactose in 30mM phosphate buffer at pH 7

24、.0and 37C.Dry bakers yeast was added to a nal concentra-tion of 25g/L,to maximize the yields by the removal of excess glu-cose.In that way,a lactotrehalose concentration of 200mM could be obtained after 10days.The product was then puried in three simple steps.First,the residual trehalose was degrade

25、d to glucose by treatment with E.coli trehalase at 37C.Next,bakers yeast was again added to consume the monosaccharides glucose and galact-ose at 30C.Finally,the solution was diluted ve times and further puried as described below.2.5.Purication by adsorptiondesorptionThe bioconversion solution (200m

26、l)was mixed with 10%acti-vated carbon for 5h at 303K and 200rpm.The carbon was then vacuum ltered through a Seitz lter (Werke GmbH,Germany)and transferred to a glass column (773cm).Elution was per-formed with 015%(v/v)of ethanol and the fractions containing the desired carbohydrate in pure form were

27、 pooled,ltered and concentrated using a rotavapor at 50C.2.6.Purication by ion-exchange chromatographyThe bioconversion solution (200ml)was loaded on an equal bed volume of Dowex 1X8(acetate form,Alfa Aesar,Germany)in a glass column (773cm)at room temperature.The resin was washed with two bed volume

28、s of deionized water,and fractions were collected for analysis.Regeneration of the resin with three bed volumes of potassium acetate (1M)was necessary after each cycle,as the resin was in a fully dissociatedstate.Table 1Physicochemical properties of the activated carbon CPG-LF.PropertiesValues Avera

29、ge particle size (mesh)1240Particle density (kg/m 3)820Average particle diameter (mm) 1.21.4Moisture content (%)3BET surface area (m 2/g)950Hardness number95162 C.Chen et al./Separation and Purication Technology 96(2012)1611672.7.Analytical procedureLactotrehalose,trehalose and other contaminants we

30、re deter-mined by HPLC(Varian Prostar)equipped with an Aminex HPX-87H column(300mm7.8mm,BIO-RAD)and an RI-detector (Varian Prostar model350).Elution was performed with5mM of sulfuric acid at65C.3.Results and discussion3.1.Adsorption isothermsAdsorption behavior is always described by the relationshi

31、p be-tween the adsorbed amount(q e)on the surface and the solute con-centration(C e)in the liquid phase,which can be described by the appropriate isotherms.For dilute aqueous solutions,such as bio-catalytic conversion mixtures,the Langmuir Eq.(3)and Freundlich Eq.(4)are preferentially used.q e qLK L

32、 C e1K L C e3q e K F C Ne4where q L and K L are the Langmuir equilibrium constants related to maximum monolayer capacity and energy of adsorption,respec-tively;K F and N are the Freundlich constants related to adsorption capacity and adsorption intensity.The Langmuir equation was orig-inally derived

33、 from the gassolid phase adsorption of activated car-bon,based on monolayer adsorption on ideally homogenous surfaces22.The Freundlich equation,in turn,was proposed to de-scribe the adsorption process on heterogeneous surfaces within an intermediate pressure range22.In this work,the adsorption of tr

34、ehalose and galactitol on acti-vated carbon has beentted to both equations,tond the most accurate isotherm for future predictions(Fig.2).It was found that the mixture6.In addition,the N values of trehalose and galactitol are favorable for adsorption,although these low numbers seem to indicate that t

35、he surface of the activated carbon CPG-LF is some-what heterogeneous(Table2).3.2.Heterogeneity of activated carbonFurther proof of the heterogeneous nature of the CPG-LF surface has been obtained by distribution studies.More specically,the solidliquid distribution coefcient K D of trehalose was calc

36、ulated from the following equation:K DC s=C w5where C s is the concentration of adsorbent in solid particles and C w is the concentration of adsorbent in aqueous solution.Its value was found to change when the amount of activated carbon was in-creased,while keeping the trehalose concentration as wel

37、l as pH and T constant(Fig.3).This implies that the surface of CPG-LF is heterogeneous,since the K D value at a homogeneous surface and xed pH should not change with the amount of adsorbent23. Moreover,a higher adsorbent dose was found to lower the q e value (Fig.3),which can be explained by the sur

38、face site heterogeneity model23.At a low dose,all types of sites are entirely exposed and the adsorption on the surface is saturated faster,resulting in a higher q e value.At a higher dose,in contrast,the availability of higher energy sites decreases with a larger fraction of lower energy sites occu

39、pied,resulting in a lower q e value.3.3.Adsorption kineticsBeing able to predict at what rate adsorption will proceed under a given condition is crucial for many applications.For that goal, various kinetic models have been proposed,ranging from pseu-do-rst order adsorption,over pseudo-second-order a

40、dsorption to intraparticle diffusion models.Of these,the pseudo-second-or-der rate expression has been widely applied to the adsorption of substrates from an aqueous solution on adsorbents like activated carbon and clay24.It is described by the following equation:qtkq2et1kqet6where q e(mmol/g)is the

41、 calculated amount of adsorbate per gram of adsorbent,and k(g/(mmol h)is the pseudo-second-order rate constant.The advantage of this model is that the initial adsorption rate can be determined from the equation without knowing any parameter beforehand.For lactotrehalose and galactitol,the adsorp-tio

42、n capacities calculated from Eq.(6)were found to be in good agreement with those determined experimentally(Table3).The pseudo-second-order model thus seems to accurately describe these compoundsadsorption on CPG-LF(Fig.4),and also indicates that these adsorption processes involve chemisorption in ad

43、dition to physisorption.Fig.2.Adsorption isotherms of trehalose(C0=40mM)and galactitol(C0=25mM).Table2Parameter valuestting isotherm models.Isotherm models Trehalose GalactitolLangmuir equationq L0.62840.7033 K L0.38860.0854 R-square0.94130.9696Freundlich equationK F0.21960.0815 N0.30040.5751 R-squa

44、re0.98720.9903C.Chen et al./Separation and Purication Technology96(2012)161167163In order to identify the mechanisms affecting the kinetics in the adsorption process,the intraparticle diffusion model was em-ployed25.It is described by the following equation:qtk pi t1=2C i7where k pi(mmol/(g h1/2)is

45、the intraparticle diffusion rate constant at stage i,C i is the intercept at stage i and is related to the thickness of the boundary layer.The plot of q t versus t1/2should present a straight line with k p as slope and C as intercept.In our case,how-ever,the model results in multi-linearity(Fig.5),r

46、evealing that two steps are involved in the process.Therst,sharper stage could be attributed to external surface adsorption,whereas the second stage could describe gradual adsorption,where intraparticle diffu-sion is a rate-limiting factor26.For both compounds,k p1is greater than k p2but C1lower tha

47、n C2(Table4).Most likely,adsorption is faster in the beginning because of the free surface area that is avail-able on the activated carbon.When the adsorbed carbohydrates have formed a thick layer,the adsorption capability slows down and becomes controlled by the transport rate of the adsorbate from

48、 the exterior to the interior sites of the carbon particles.The fact that the plots do not pass through the origin indicates that intraparticle diffusion is only one of the rate-controlling steps in the adsorption process.Some other adsorption mechanism could be involved,such as ion exchange,precipi

49、tation or other physical phenomena27.adsorbent dose on the distribution coefcient(K D)and adsorption capacity(q e)of trehalose(40mM)atTrehalose Galactotitol40250.360.2018.838.10.360.210.9970.998Fig.4.Adsorption kinetics of trehalose(C0=40mM)and galactitol(C0=25mM)at 303K,using a sorbent dose of10%(m

50、/v).Fig.5.Intraparticle diffusion model for the adsorption of trehalose(C0=40mM) and galactitol(C0=25mM)on activated carbon(10%)at303K.C.Chen et al./Separation and Purication Technology96(2012)161167165Table4Values for the parameterstted with the intraparticle diffusion model.Substrates Initial conc

51、entration(mM)k p1k p2C1C2R1-square R2-squareTrehalose400.1360.0100.1710.3270.9620.971 Galactotitol250.06740.0360.1090.1920.9740.909Fig.6.Anion exchange separation prole of the bioconversion mixture.bioconversion mixture from activated carbon,after use of ion exchange(gradient elution with5%ethanol f

52、rom1X8resin and washed with deionized water.Logically,the uncharged sugars and alcohols did not bind to matrix while the negatively charged(glycosyl)phosphate and lactate were retained (Fig.6).Theow-through(containing acetate as counterion from the resin)was then mixed with activated carbon and shak

53、en for 5h to reach the adsorption equilibrium.The adsorbed amounts of lactotrehalose,galactitol,and glycerol were found to be76,35 and19%,respectively.This shows that lactotrehalose is the most preferable compound to bind with the activated carbon,and that galactitol is the main contaminant in the a

54、dsorption process.How-ever,selective desorption with aqueous ethanol as eluent allowed to purify the lactotrehalose even further.Indeed,separation of mono-and disaccharides can be accomplished by elution with an ethanol gradient,which gradually removes the stronger binding components20.In our case,m

55、ost of the galactitol and glycerol was already removed by washing with water,whereas the lactot-rehalose only eluted at an ethanol concentration of10%or higher (Fig.7).In that way,lactotrehalose could be recovered with a yield of31%and a purity of over97%.As an alternative,the direct purication of l

56、actotrehalose from the pretreated bioconversion mixture was evaluated,thus omitting the ion-exchange chromatography.In the adsorption step,a similar pattern was observed as in the previous procedure,with a binding preference in the order of lactotrehalosegalactitolglycerol acetatelactatephosphate.Li

57、kewise,selective desorption of lac-totrehalose with1015%ethanol resulted in a yield of24%and a purity of more than97%(Fig.8).The somewhat lower yield of lac-totrehalose in this one-step procedure can probably be explained by the burden of ions in the starting mixture.Nevertheless,the product is high

58、ly pure and the omission of the costly chromato-graphic step is benecial from a commercial point of view.4.ConclusionsThe sorption equilibrium and kinetics of trehalose and galactitol on activated carbon CPG-LF1240were determined experimen-tally.The adsorption capacity was found to be higher for trehalose than for galactitol,which could be accurately described by the Freundlich equation.The adsorption kine