Cross-linking enzyme aggregates in the macropores of silica gel: A practical and efficient method for enzyme stabilization
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Biochemical Engineering Journal 52 (2010) 168174
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Biochemical Engineering Journal
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Cross-l poA pract ab
Mengfan eState Key Labo n Univ
a r t i c l
Article history:Received 18 NReceived in reAccepted 8 Au
Keywords:EnzymeImmobilizatioCLEAs (cross-lMacroporous sDipeptide synPapain
painilityandzymeture oo freever, tEAs-ield
Enzymes are biocatalysts with high specicity and efciencywhich are ining, pharmacells [1,2].often limitestability. Inimmobilizephysical adsulation wi
In resenlinked enzattention. Bare of supralized with hof the undeusually beloCLEAs fromespecially was the CLEAmight lead tresistanceo
solution and thereby cause low catalytic efciency . These resultin some problems for the broad use of CLEAs.
In order to solve these problems, some new reactor styles were
1369-703X/$ doi:10.1016/j.creasingly applied in chemical synthesis, food process-ceuticals, proteomic analysis, biosensors, and biofuelHowever, the widespread application of enzymes isd because of their poor reusability and low operationalorder to improve enzyme properties, many carrier
d enzyme strategies have been researched includingsorption, multipoint covalent attachment and encap-th organic or inorganic materials .t years, a new immobilized enzyme strategy, cross-yme aggregates (CLEAs), has attracted increasingy cross-linking the physical enzyme aggregates whichmolecular structures, enzyme can be simply immobi-igh stability and high volume activity. However, onesirable properties of CLEAs is that their particle size isw 10m [6,7]. Thus, it is difcult to isolate and recoverthe reaction system only by centrifugation or ltrationhen the substrate particles are in the same size ranges. Moreover, centrifugation and ltration treatmentso forming increasedclumpsdue to the lowcompressionfCLEAs,whichwouldhamperCLEAs todisperseagain in
ding author. Tel.: +86 22 2740 7799; fax: +86 22 2740 7599.ress: email@example.com (W. Qi).
designed based on the properties of CLEAs. Cabana et al.  devel-oped a novel perfusion basket reactor (BR) which consisted ofan unbafed basket lled with laccase CLEAs suspension and amarine propeller in it. Continuous agitation avoided CLEAs form-ing clumps and kept their particles dispersed well, which thereforeretained CLEAs with high activity and stability after 7-day oper-ation period. Besides, more and more approaches were focusedon the combination of CLEAs technology with traditional immo-bilization methods. Hilal et al.  prepared CLEAs inside thepores of microltration membranes and applied these biocatalyticmembranes to cross-ow membrane reactor. Wilson et al. encapsulated CLEAs of penicillin G acylase (PGA) into a very rigidlens-shaped polyvinyl alcohol (PVA) hydrogel particles (LentiKats)and successfully improved the inadequate mechanical propertiesof CLEAs. Kim et al.  entrapped CLEAs of -chymotrypsinand lipase in the mesocellular pores of the hierarchically-orderedmesocellular mesoporous silica (HMMS) to form a ship-in-a-bottlestructure which avoided CLEAs leaching out of HMMS through nar-rowmesoporous channels (13nm).However, this approachneededthe special HMMS carrier, and the mesocellular pore size was only37nm which limited the maximum loading of CLEAs to a certaindegree.
To overcome these problems, we developed a simple strategyfor preparing a new kind of CLEAs through one-step-cross-linking
see front matter 2010 Elsevier B.V. All rights reserved.bej.2010.08.003inking enzyme aggregates in the macroical and efcient method for enzyme st
Wang, Wei Qi , Qingxin Yu, Rongxin Su, Zhimin Hratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianji
e i n f o
ovember 2009vised form 16 July 2010gust 2010
ninked enzyme aggregates)ilica gel
a b s t r a c t
Cross-linked enzyme aggregates of paMSG) in order to improve the operabfrom simple adsorption, precipitationble structure that did not leak out enCLEAs and MSG. The optimal tempera7.0, whichwere improved compared tstability and thermal stability. Moreosize and active properties. By using CLGln synthesis was achieved with the yas biocatalyst.om/ locate /be j
res of silica gel:ilization
ersity, Tianjin 300072, PR China
were prepared in commercial macroporous silica gel (CLEAs-and mechanical stability of CLEAs. CLEAs-MSG was obtainedone-step-cross-linking. CLEAs-MSG was characterized by sta-from the macropores because of covalent bonding betweenf papain CLEAs in MSG was 4090 C and the optimal pH waspapain and CLEAs. The CLEAs-MSG also enhanced the storagehe CLEAs-MSG exhibited good reusability due to its suitableMSG of papain as biocatalyst, the kinetically controlled z-Ala-of 32.9%, which was almost equal to that by using free papain
2010 Elsevier B.V. All rights reserved.
M. Wang et al. / Biochemical Engineering Journal 52 (2010) 168174 169
enzymes into the pores of commercial macroporous silica gel(CLEAs-MSG). The macroporous silica gel (MSG) is usually usedas the catalyst carrier and the packing for HPLC column. Papain(E.C. 184.108.40.206), a thiol protease, was employed as a model enzymebecause ofas in the thpeptides [1step-cross-optimal catCLEAs-MSGysis, the CLEof N-(benzya precursornutrition.
Papain (China). MSGLtd. (Qingd(z-Ala-OMeLtd. (Shanghtaraldehydegrade andother puric
The amivious pape(1:1), washThen 4g of(APTES/ethvacuum attilled waterthe composments werefrom 1400100mg premined by sa
The CLEIn a 50mL cpapain in bupH 7.0)wasring,GAwawas dilutedpelletwaswin 0.2mol/L
CLEAs-Mmodied M(0.15g/mL,3.5h at 25
(0.2mol/L pshaking totion,GAwaof 2% andwashed forphosphateCLEAs-MSGtion time,cross-linkin
experiment, i.e. only one factor value was varied to determine theenzyme activity when the other factors were xed.
2.4. Enzyme activity assay
actiby tn (1%vatioddedzym
D275e (me (
optieren uninednt pHe sam
howallyd wl wejustesis rits versatility in hydrolysis reactions [13,14] as wellermodynamically or kinetically controlled synthesis of517]. The effects of adsorption, precipitation and one-linking on the enzyme activity were investigated. Thealytic temperature and pH as well as the stability ofwere measured. To verify the practicability in biocatal-As-MSG of papain was applied to the kinetic synthesisloxycarbonyl)-alanyl-glutamine (z-Ala-Gln), which isof an importantly functional dipeptide (Ala-Gln) for
ls and methods
16,000U/g) was purchased from Pangbo Ltd. (Guangxi,(with diameter of 5mm) was purchased from Meigao
ao, China). N-(benzyloxycarbonyl)-alanyl-methylester) and glutamine (Gln)werepurchased fromGLBiochemai, China). 3-Aminopropyltriethoxysilane (APTES), glu-(GA, 50%, w/v) and other reagents were of analytical
btained fromcommoncommercial sourceswithout fur-ation.
no-modied MSG was prepared as described in a pre-r . MSG was pre-puried with hydrochloric acided with distilled water, and dried at 150 C for 12h.these dried MSG were added to the modifying regentanol, 1/2, v/v) and the ethanol was evaporated under60 C. The amino-modied MSG was washed with dis-for 3 times and dried at 100 C. In order to characterizeing groups of modied MSG, the IR spectrum measure-performed on a Nicolet Nexus 870 FT-IR spectrometerto 400 cm1 with 1mg sample powder dispersed inssed KBr discs. The content of amino-group was deter-licylaldehyde .
tion of CLEAs and CLEAs-MSG of papain
As of papain were prepared by conventional method.entrifuge tube with a magnetic stirrer bar, 1mL of freeffer solution (5mg/mL, 0.2mol/L phosphate buffer, andadded into 9mL of ethanol at 25 C. After 30min of stir-s addedandstirred for12hat25 C. Then the suspensionwith 10mL phosphate buffer and centrifugated. Theashed for3 timesbyphosphatebuffer andnally storedphosphate buffer (pH 7.0) at 4 C.SG of papain was prepared as follows. The amino-SG was mixed with free papain in buffer solution0.2mol/L phosphate buffer, pH 7.0) and shaken forC. Then the samples were washed by aqueous bufferhosphate buffer, pH 7.0) and added into ethanol withprecipitate the free papain for 10min. After precipita-s added into the suspensionup to thenal concentrationshaken for 4h. After cross-linking, CLEAs-MSG was3 times by phosphate buffer and stored in 0.2mol/Lbuffer (pH 7.0) at 4 C. The preparing conditions ofincluding buffer pH, enzyme concentration, adsorp-
precipitant type, cross-linker (GA) concentration andg time have all been optimized through single factor
Theminedsolutioof actiwere ation, entrichloreactiofor 10of the sof enzy
Stowere dphate band CLtilled wfree en
Thewere dphatefor10mCLEAs-at each
As skineticdistilleethanoand adsynthevity of free enzyme, CLEAs and CLEAs-MSG were deter-he hydrolysis of casein. In a 10mL tube, 1mL of casein, w/v) in 0.1mol/L phosphate buffer (pH 7.0) and 1mL
n solution (contained 20mmol cys and 1mmol EDTA)and incubated at 35 C for 10min. After the incuba-
e sample was added. After another 10min, 2mL of 10%tic acid solution was added into the tube to quench thee mixed solution was incubated with stirring at 35 Cand then ltered. The spectrophotometric absorbancenatantwasmeasured at 275nm. The hydrolytic activitycan be calculated as formula (1) described:
10 = U/mg (1)
is the absorbance value at 275nm; Ew is the amount ofg); 0.116 is the absorbance value at 275nm of 1molmol1), and 10 is the reaction time (min).
l conditions for enzyme activity
mal temperature of the free enzyme, CLEAs and CLEAs-determined by adding the enzyme into the substrateder different temperatures, and the optimal pH wasby adding the enzyme into the substrate solution of. The activities of these immobilized enzyme and freeples were determined.
stabilities of the free enzyme, CLEAs and CLEAs-MSGined by incubating enzyme samples in 0.2mol/L phos-
r (pH 7.0) without substrate at 4 C. Every 3 days, CLEAsMSGwere separated from the buffer andwashed by dis-. Then the activities of these immobilized enzyme ande samples were determined.
stabilities of the free enzyme, CLEAs and CLEAs-MSGined by incubating enzyme samples in 0.2mol/L phos-
r (pH 7.0) without substrate at 40, 50, 60, 70, and 80 Cfter incubation, theactivitiesof freeenzyme,CLEAsandwere assayed immediately. Then the residual activitiesperature were determined.
s-MSG was separated by lter-paper from the reactionr the activity assay, and then washed with 0.2mol/Lbuffer (pH 7.0) for 3 times to determine the activity
ally controlled synthesis of z-Ala-Gln
n in Fig. 1, z-Ala-Gln was enzymatically synthesized bycontrolled method. 2.193g (15mmol) of Gln in 30mLater and 0.3536g (1.5mmol) of z-Ala-OMe in 1.5mLre added into a 50mL ask with stir at 35 C, 160 rpmd to pH 9.5 with sodium hydroxide. Then the peptideeaction was carried out by adding 2400U free papain
170 M. Wang et al. / Biochemical Engineering Journal 52 (2010) 168174
Fig. 1. The reaction scheme of kinetically controlled z-Ala-Gln synthesis.
or 2400U CLEAs-MSG, respectively. The reaction process of pep-tide synthesis and the quantitative analysis of the product wereperformed by HPLC under the following conditions: YMC-PackODS-A C18 column (column i.d. 4.6mm150mm); isocratic elu-tion (35% acetonitrile in water with 0.1% triuoroacetic acid; owrate 1mL/min; monitor at 214nm). The yield of z-Ala-Gln was cal-culated as formula (2):
Y (%) = cs0 c c
where cs0 aAla-OMe, rez-Ala-OH.
All the r
In orderon the poroAPTES becaally used asmethod, noform the CLto silica gelperformedobserved atat 3400 cm1100 cm1.while it couindicated th
3.2. Preparation of CLEAs-MSG
The preparation of CLEAs-MSG consisted of three simple stepsas shown in Fig. 2. In the rst step, free papains were embeddedin the modied MSG through diffusing into the inner channels andadsorbing on the porous surface; in the second step, enzyme aggre-gates were formed in channels and on the inner surface of MSGthrough precipitating; and in the nal step, CLEAs were formed
Enzymorpton od benter shof the w
3a, thbe the actiivityindiction.mL,waturaeon
sdiffe ince doms ofer cs h
cs0 100 (2)
nd cs were the initial and terminal concentration of z-spectively; and ch was the terminal concentration of
esults in this paper were performed in triplicates.
modication of MSG for CLEAs-MSG preparation
to stabilize CLEAs in MSG, the silanol groups presentus surface of MSG were modied into amino-groups byuse these amino-groups can reactwithGAwhich is usu-a bifunctional agent for enzyme cross-linking. By this
t only enzymeaggregateswere cross-linking together toEAs, but also the formed CLEAs were covalently bondedat the same time. The IR spectrum measurements werefor the modied MSG. The band assigned to NH2 was1600 cm1; the band assigned to NH was observed
1; and the band assigned to CN was observed atThe content of amino-group was 8.5103mol/mg,ld not be determined before modication. These resultsat amino-modied MSG was formed.
bilizatimethothe outhe pHtion tim[21,22was stuin Fig.mightand ththeactFig. 3bcentra0.15g/were stion timpapainwith thwas thsurfacethe innFig. 2. Schematic illustration of the CLEAs-MSG pretly bonded to the MSG through one-step-cross-linking.
e adsorptionion is one of the simplest methods for physical immo-f enzyme since its low cost and mild process . Thisets from interactions between the carrier surface andell of the enzyme. These interactions can be altered bye solution, the enzyme concentration and the adsorp-hich might affect the adsorption capacity of the carriertly, the effect of pH on the activity in adsorption stepusingpapain solutions of different pHvalues. As showne highest activity was found to be at pH 6.07.0 whiche compromise point of the adsorptive capacity of MSGve property of papain. Secondly, in addition to pHvalue,alsodependedon the concentrationof enzymesolution.ated that activity rosewith the increase of enzyme con-No signicant increasing in activitywas observed abovehich indicated that the enzymes embedded in theMSGted at this concentration. Thirdly, the effect of adsorp-activitywas shown inFig. 3c. Before3h,moreandmoreused into the inner channels andwereadsorbedonMSGreasingof time. Thus theamountof embeddedenzymesinant factor for activity. Between 3 and 4h, the inner
MSG have been saturated by adsorbed enzymes andhannels have also been saturated by soluble enzymes.paration process.
M. Wang et al. / Biochemical Engineering Journal 52 (2010) 168174 171
Fig. 3. The effadsorption timgure as 100%3h at 25 C; (b7.0 enzyme so
Thus the acphase. But achannel becmore and mble enzymedominant f
Therefore, the adsorption time was controlled at about 3.5h toobtain the highest activity.
Enzyme precipitatione enzymes had been embedded in the MSG after adsorp-hen precipitants were added, enzyme aggregates formednging the properties that effect the proximity of the freees.Usually, theprotein recovery agents, suchas salts, organicts, non-ionic polymers or acids can be used as precipitants.3.2.2.Fre
tion. Wby chaenzymsolvenects of (a) enzyme solution pH; (b) enzyme concentration; and (c)e on the relative activity. Assuming the highest activity value of each. Adsorption conditions: (a) 0.2 g/mL enzyme solution incubated for) pH 7.0 enzyme solution incubated for 3h at 25 C; (c) 0.2 g/mL, pHlution incubated at 25 C.
tivity of CLEAs-MSG achieved the highest value in thisfter 4h, the autohydrolysis of soluble enzymes in MSGame seriouswith the increasing of time .Moreover,ore adsorbed enzymes were hydrolyzed by the solu-
s [24,25]. Thus the autohydrolysis behavior became theactor for the rapid decrease of activity in this phase.
Fig. 4 showed four precipitants and their residual activities afterprecipitation process. It can be observed that precipitating freepapains with anhydrous ethanol yielded almost 100% residualactivity compared with equivalent free enzyme, but less than 20%residual activitywas recoveredwithmethanol. This result indicatedthat different precipitants played different role on the activity forthe same enzyme. Through precipitation, the enzyme aggregateswere held together by noncovalent bond and formed supramolec-ular structures which might keep the stable conformation xed.
3.2.3. One-step-cross-linkingWith one-step-cross-linking by GA, CLEAs were formed in the
macropores and covalently bonded on the inner surface of modi-ed MSG through Schiff base reaction. The effects of cross-linkingtime and GA concentration on the activity of CLEAs-MSG wereshown in Fig. 5. It can be seen that either increasing GA concen-tration or prolonging the cross-linking time might decrease theactivity ofwhich wasfor enzymelinking reacconcentratiof enzymeundesired eand amongdesired oneTherefore, wthe reactioity of CLEAsattached tobonds wereores even twas preparwas incuba
Fig. 4. The effout precipitatammonium suenzyme-adsorCLEAs-MSG. These resulted from the property of GAnot only a cross-linking agent but also a denaturant. The active conformation might be ruined if the cross-tion affected the active sites of enzyme. The high GAon and the long reaction time might intensify the lossactivity. Moreover, at the high GA concentration thexcessive self-cross-linking among enzyme aggregatesMSG surfaces would compete the effective sites for the-step-cross-linking between CLEAs and MSG surfaces.e controlled the GA concentration to be 2% and limited
n time to be 4h in order to obtain the highest activ--MSG.In a word, by one-step-cross-linking, CLEAs werethe inner surface ofMSG through covalent bonds. Theserm enough to avoid CLEAs leaking from the macrop-
hough the pores were huge to CLEAs. Here, CLEAs-MSGed under the optimal conditions: 1 g of modied MSGted in the 0.15g/mL and pH 7.0 enzyme solution for
ect of precipitants on the papain activity. Assuming the activity with-ion as 100%. Precipitation conditions: 4mL precipitant (saturatedlfate, anhydrous alcohol, 100% acetone or 100% methanol) per grambed MSG, pH 7.0 and 25 C.
172 M. Wang et al. / Biochemical Engineering Journal 52 (2010) 168174
Fig. 5. The effects of cross-linking time and GA concentration on the activity ofCLEAs-MSG. Assuming the CLEAs-MSG activity value under 2% GA concentrationand 4h cross-linking as 100%.
3.5 h at 25 C, and then precipitated with 4mL anhydrous alcoholand cross-linked with 2% GA for 4h. The resulting activities were960U/g CLEAs-MSG and 14,000U/g CLEAs.
3.3. Structural characterization by SEM
The SEMshown in Fand d, respof 5000) tenough forlimitation a(magnicatores as CLEnot leak outhrough onously obserthe surface
Fig. 7. The optimal temperature curves of free and immobilized enzyme. Assumingthe highest activity of free enzyme, CLEAs and CLEAs-MSG as 100%, respectively.
rough with CLEAs bonded on it (Fig. 6d). Additionally, the solidporous structure provided the frame for CLEAs to be accommo-dated. Therefore, the size of CLEAs-MSG was large enough to makebiocatalysts more suitable in industrial application. Moreover,CLEAs-MSG could avoid forming CLEAs clumps after centrifugationand ltration because of the supporting effect of MSG.
The ooptiAs a
mperIt couder tprov
Fig. 6. SEM imimage of modied MSG before immobilization wasig. 6a and c, and the CLEAs-MSG was shown in Fig. 6bectively. It could be observed in Fig. 6a (magnicationhat the pores and channels of modied MSG were largefree enzymes to pass through without much diffusionnd for CLEAs to be accommodated. As shown in Fig. 6bion of 5000), CLEAs were formed in these macrop-As-MSG, and the CLEAs entrapped in the pores wouldt because the CLEAs were covalently bonded to MSGe-step-cross-linking. This attachment could be obvi-ved in Fig. 6c and d (magnication of 20,000) thatof the MSG was very smooth (Fig. 6c) but became
3.4. Thand CL
for CLEmonlyfree enmal terange.in a wiThis images of (a) modied MSG magnied 5000; (b) CLEAs-MSG magnied 5000; (c) moditimal biocatalytic conditions of free enzyme, CLEAsSG
ptimal temperaturemal temperature was at 80 C for free papain, 5080 Cnd 4090 C for CLEAs-MSG as shown in Fig. 7. Com-obilization can increase the optimal temperature ofe. However, CLEAs-MSG did not increase the opti-ature obviously but extended the optimal temperatureldbe seen that CLEAs-MSGmaintained thehigh activityemperature range compared to free papain and CLEAs.ement resulted from the covalent cross-linking of GAed MSG magnied 20,000; (d) CLEAs-MSG magnied 20,000.
M. Wang et al. / Biochemical Engineering Journal 52 (2010) 168174 173
Fig. 8. TheoptimalpHcurvesof freeand immobilizedenzyme.Assuming thehighestactivity of free enzyme, CLEAs and CLEAs-MSG as 100%, respectively.
among enzymes and between CLEAs and MSG surfaces. This struc-ture stabilized the conformation of active sites of enzyme, whichcould be easily ruined by the change of reaction temperature.
3.4.2. The optimal pHAs show
CLEAs-MSGThis shift mbasic aminoactive site oCLEAs-MSGpH 4.0 comthat CLEAs-harsh cond
3.5. The sta
3.5.1. StoraTo inves
bility of pap4 C in aqueits total actretained ab
Fig. 9. The stAssuming the
Fig. 10. The thatures. Assumas 100%. Incub
35% after 15nosignicathat of CLEA
. It isnd CLor ene int
Thermtheinedtemp10. Itits inwhicout 7lly (esulette
covaldownther word, it was more difcult to ruin the conformation ofn in Fig. 8, the optimal pH values of free papain andwere observed as 7.0, but that of CLEAs was as 6.0.ight be caused by the ionization changes of acidic oracid side chains in the microenvironment around thef enzyme or on the surface group of MSG . Besides,retained the higher activity above pH 7.0 and belowpared with free enzyme and CLEAs, which indicatedMSG protected papain from intensively deactivating atitions.
bility of free enzyme, CLEAs and CLEAs-MSG
ge stabilitytigate the effect of immobilization on the storage sta-ain, free enzyme, CLEAs and CLEAs-MSG were stored atous buffer. As shown in Fig. 9, free enzyme lost almostivity after only 3 days, but CLEAs and CLEAs-MSG bothout 50% of their initial activities after 3 days and about
determferentin Fig.83% ofbationwas abmaticaThese rmuchbof CLEinter-cthe covthesebreakIn anoorage stability of free and immobilized enzyme in aqueous buffer.initial enzyme activity before incubation as 100%.
enzyme inwere under
3.6. The rec
As menttance of CLindustrial The CLEAs-the size coThe propercatalyst frorecovered a100%, 97.3%the high acthat CLEAsapplicationermal stability of free and immobilized enzymes at different temper-ing the initial enzyme activity (determined at 35 C) before incubationation conditions: pH 7.0 aqueous buffer, incubated for 10min.
days. These results demonstrated that CLEAs-MSG hadntenhancementon the storage stability comparingwiths but dramatically improved the store stability of freebecause that the multipoint immobilization of CLEAs-EAs prevent the dissociation of enzyme from its matrixzyme aggregates) and further prevent the leaking ofo aqueous buffer.
al stabilityrmal stability of free and immobilized papain wereby incubating them in aqueous buffer (pH 7.0) at dif-eratures for 10min. The residual activities were givencould be seen that the CLEAs-MSG retained more thanitial activity at each temperature after 10min of incu-hwas a little higher than that of CLEAs (residual activity080%), but the free papain lost its initial activity dra-
residual activity was about 2050%) after incubation.ts indicated that the thermal stability of CLEAs-MSGwasr than that of free enzymeandCLEAs. This enhancementSG in thermal stability might be due to the covalentlinking between CLEAs and MSG surfaces as well ast intra-cross-linking among enzyme aggregates. Due toent bonds, CLEAs-MSG needed much more energy tothe active conformation than free enzyme and CLEAs.CLEAs-MSG than that of free enzyme and CLEAs if theythe same thermal condition.
overy of CLEAs-MSG
ioned above, the small size and low compressive resis-EAs made it difcult to recover and apply to someelds. However, CLEAs-MSG overcame these problems.MSGpelletswere of 5mmdiameter asweprepared, anduld be also adjusted according to the practical needs.size of CLEAs-MSG facilitated the separation of bio-m reaction system only by ltration. The initial andctivities of CLEAs-MSG for each determination were, 89.8% and 76.1%. That meant CLEAs-MSG still retainedtivity after using for 4 times. These results indicated-MSG improved the properties of CLEAs in practical.
174 M. Wang et al. / Biochemical Engineering Journal 52 (2010) 168174
Fig. 11. The ticonditions: 0.5enzyme.
3.7. The appsynthesis of
Since thapplied in kin Fig. 11, tby CLEAs-Mequal units32.9% at 14using free ehad the equcontrolled stion equilibstability ofand purica
A simplepores of siliof MSG werCLEAs fromstrategy. Itconcentratition and cractivity in tof papain dconditions,stability thasimilar to frGln with thThe convenof CLEAs-Mefcient solcation.
The authNew Centu0386), the K
the 863 Program of China (2008AA10Z318), the Natural Sci-ence Foundation of China (No. 20976125, No. 31071509), theScience Foundation of Tianjin (No. 10JCYBJC05100)), and the Pro-gram of Introducing Talents of Discipline to Universities of China(No. B06006).
covaletechnKallen: the. Abduonellosopor. SheldEAs):catal.ao, Im226choevtwijkes of c762aban
a perfruptinHilal,regate04) 13ilsonapsulion of562. Kim,te, P.Wierarcyme s. Wupropealtra. Sekutially. Chenhasictehle,s: a nhnol.chellesynth
nce, Sm onl. Che. Wadme course in kinetically controlled synthesis of z-Ala-Gln. Reactionmol/L z-Ala-OMe, 0.05mol/L Gln, 35 C, pH 9.5, 160 rpm and 2400U
lication of CLEAs-MSG to kinetically controlledz-Ala-Gln
e excellent stability and recovery, CLEAs-MSG wasinetically controlled synthesis of z-Ala-Gln. As shownhe time course in synthetic performance of z-Ala-GlnSG was quite similar to that by free enzymes when theof activity (2400U) were added. The nal yield was
0min by using CLEAs-MSG as biocatalyst and 31.9% bynzyme. This result indicated that CLEAs-MSG not onlyivalently catalytic ability to free enzyme in kineticallyynthesis of z-Ala-Gln, but also do not change the reac-rium and the selectivity of papain. In addition, the highCLEAs-MSG in aquatic solvents facilitated the isolationtion of reaction products.
method for preparing CLEAs of papain in the macro-ca gel (CLEAs-MSG) was proposed. The inner channelse large enough to accommodate CLEAs and preventedleaking out effectively by the one-step-cross-linking
was demonstrated that pH of enzyme solution, enzymeon, adsorption time, precipitant type, GA concentra-
 A. Sbio
 H.Ein i
 P. WbyBio
 L. C217
 R. SRantur754
 H. Cindis
 M.IGrain henz
 P. StideTec
 V. Stide
 H. ImiuAna
oss-linking time had signicant effects on CLEAs-MSGhe process of preparing CLEAs-MSG. The CLEAs-MSGisplayed the improved optimal temperature and pHand exhibited the higher storage stability and thermaln free papain and in some cases CLEAs. Additionally,ee papain, CLEAs-MSG catalyzed the synthesis of z-Ala-e yield of 32.9% through kinetically controlled method.ient preparing process and the excellent propertiesSG reported in this paper may provide a feasible andution to the drawbacks of enzymes in industrial appli-
ors thank the nancial support from the Program forry Excellent Talents in Chinese University (NCET-08-ey Project of Chinese Ministry of Education (108031),
surface b J.M. Palom
J.M. Guis(octadecythe open
 H. Takahenzymesstability a4445 (2
 J. Felipe DJ. Mol. Ca
 B.C. Kim,Petritis, Dstable trydigestion
 A. Berlin,lignin-binhydrolysi
 B. Yang, DAvicel ce
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Cross-linking enzyme aggregates in the macropores of silica gel: A practical and efficient method for enzyme stabilizationIntroductionMaterials and methodsMaterialsSurface modificationPreparation of CLEAs and CLEAs-MSG of papainEnzyme activity assayOptimal conditions for enzyme activityStorage stabilityThermal stabilityRecovery stabilityKinetically controlled synthesis of z-Ala-Gln
Results and discussionSurface modification of MSG for CLEAs-MSG preparationPreparation of CLEAs-MSGEnzyme adsorptionEnzyme precipitationOne-step-cross-linking
Structural characterization by SEMThe optimal biocatalytic conditions of free enzyme, CLEAs and CLEAs-MSGThe optimal temperatureThe optimal pH
The stability of free enzyme, CLEAs and CLEAs-MSGStorage stabilityThermal stability
The recovery of CLEAs-MSGThe application of CLEAs-MSG to kinetically controlled synthesis of z-Ala-Gln