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Biochemical Engineering Journal 52 (2010) 168–174

Contents lists available at ScienceDirect

Biochemical Engineering Journal

journa l homepage: www.e lsev ier .com/ locate /be j

ross-linking enzyme aggregates in the macropores of silica gel:practical and efficient method for enzyme stabilization

engfan Wang, Wei Qi ∗, Qingxin Yu, Rongxin Su, Zhimin Hetate Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China

r t i c l e i n f o

rticle history:eceived 18 November 2009eceived in revised form 16 July 2010ccepted 8 August 2010

a b s t r a c t

Cross-linked enzyme aggregates of papain were prepared in commercial macroporous silica gel (CLEAs-MSG) in order to improve the operability and mechanical stability of CLEAs. CLEAs-MSG was obtainedfrom simple adsorption, precipitation and one-step-cross-linking. CLEAs-MSG was characterized by sta-ble structure that did not leak out enzyme from the macropores because of covalent bonding betweenCLEAs and MSG. The optimal temperature of papain CLEAs in MSG was 40–90 ◦C and the optimal pH was

eywords:nzymemmobilizationLEAs (cross-linked enzyme aggregates)acroporous silica gelipeptide synthesis

7.0, which were improved compared to free papain and CLEAs. The CLEAs-MSG also enhanced the storagestability and thermal stability. Moreover, the CLEAs-MSG exhibited good reusability due to its suitablesize and active properties. By using CLEAs-MSG of papain as biocatalyst, the kinetically controlled z-Ala-Gln synthesis was achieved with the yield of 32.9%, which was almost equal to that by using free papainas biocatalyst.

© 2010 Elsevier B.V. All rights reserved.

apain

. Introduction

Enzymes are biocatalysts with high specificity and efficiencyhich are increasingly applied in chemical synthesis, food process-

ng, pharmaceuticals, proteomic analysis, biosensors, and biofuelells [1,2]. However, the widespread application of enzymes isften limited because of their poor reusability and low operationaltability. In order to improve enzyme properties, many carriermmobilized enzyme strategies have been researched includinghysical adsorption, multipoint covalent attachment and encap-ulation with organic or inorganic materials [3–5].

In resent years, a new immobilized enzyme strategy, cross-inked enzyme aggregates (CLEAs), has attracted increasingttention. By cross-linking the physical enzyme aggregates whichre of supramolecular structures, enzyme can be simply immobi-ized with high stability and high volume activity. However, onef the undesirable properties of CLEAs is that their particle size issually below 10 �m [6,7]. Thus, it is difficult to isolate and recoverLEAs from the reaction system only by centrifugation or filtration

specially when the substrate particles are in the same size ranges the CLEAs’. Moreover, centrifugation and filtration treatmentsight lead to forming increased clumps due to the low compression

esistance of CLEAs, which would hamper CLEAs to disperse again in

∗ Corresponding author. Tel.: +86 22 2740 7799; fax: +86 22 2740 7599.E-mail address: qiwei@tju.edu.cn (W. Qi).

369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2010.08.003

solution and thereby cause low catalytic efficiency [8]. These resultin some problems for the broad use of CLEAs.

In order to solve these problems, some new reactor styles weredesigned based on the properties of CLEAs. Cabana et al. [9] devel-oped a novel perfusion basket reactor (BR) which consisted ofan unbaffled basket filled 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. [10] prepared CLEAs inside thepores of microfiltration membranes and applied these biocatalyticmembranes to cross-flow membrane reactor. Wilson et al. [11]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. [12] 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-row mesoporous channels (13 nm). However, this approach needed

the special HMMS carrier, and the mesocellular pore size was only37 nm 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

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M. Wang et al. / Biochemical En

nzymes into the pores of commercial macroporous silica gelCLEAs-MSG). The macroporous silica gel (MSG) is usually useds the catalyst carrier and the packing for HPLC column. PapainE.C. 3.4.22.2), a thiol protease, was employed as a model enzymeecause of its versatility in hydrolysis reactions [13,14] as wells in the thermodynamically or kinetically controlled synthesis ofeptides [15–17]. The effects of adsorption, precipitation and one-tep-cross-linking on the enzyme activity were investigated. Theptimal catalytic temperature and pH as well as the stability ofLEAs-MSG were measured. To verify the practicability in biocatal-sis, the CLEAs-MSG of papain was applied to the kinetic synthesisf N-(benzyloxycarbonyl)-alanyl-glutamine (z-Ala-Gln), which isprecursor of an importantly functional dipeptide (Ala-Gln) for

utrition.

. Materials and methods

.1. Materials

Papain (16,000 U/g) was purchased from Pangbo Ltd. (Guangxi,hina). MSG (with diameter of 5 mm) was purchased from Meigaotd. (Qingdao, China). N-(benzyloxycarbonyl)-alanyl-methylesterz-Ala-OMe) and glutamine (Gln) were purchased from GL Biochemtd. (Shanghai, China). 3-Aminopropyltriethoxysilane (APTES), glu-araldehyde (GA, 50%, w/v) and other reagents were of analyticalrade and obtained from common commercial sources without fur-her purification.

.2. Surface modification

The amino-modified MSG was prepared as described in a pre-ious paper [18]. MSG was pre-purified with hydrochloric acid1:1), washed with distilled water, and dried at 150 ◦C for 12 h.hen 4 g of these dried MSG were added to the modifying regentAPTES/ethanol, 1/2, v/v) and the ethanol was evaporated underacuum at 60 ◦C. The amino-modified MSG was washed with dis-illed water for 3 times and dried at 100 ◦C. In order to characterizehe composing groups of modified MSG, the IR spectrum measure-

ents were performed on a Nicolet Nexus 870 FT-IR spectrometerrom 1400 to 400 cm−1 with 1 mg sample powder dispersed in00 mg pressed KBr discs. The content of amino-group was deter-ined by salicylaldehyde [19].

.3. Preparation of CLEAs and CLEAs-MSG of papain

The CLEAs of papain were prepared by conventional method.n a 50 mL centrifuge tube with a magnetic stirrer bar, 1 mL of freeapain in buffer solution (5 mg/mL, 0.2 mol/L phosphate buffer, andH 7.0) was added into 9 mL of ethanol at 25 ◦C. After 30 min of stir-ing, GA was added and stirred for 12 h at 25 ◦C. Then the suspensionas diluted with 10 mL phosphate buffer and centrifugated. Theellet was washed for 3 times by phosphate buffer and finally stored

n 0.2 mol/L phosphate buffer (pH 7.0) at 4 ◦C.CLEAs-MSG of papain was prepared as follows. The amino-

odified MSG was mixed with free papain in buffer solution0.15 g/mL, 0.2 mol/L phosphate buffer, pH 7.0) and shaken for.5 h at 25 ◦C. Then the samples were washed by aqueous buffer0.2 mol/L phosphate buffer, pH 7.0) and added into ethanol withhaking to precipitate the free papain for 10 min. After precipita-ion, GA was added into the suspension up to the final concentrationf 2% and shaken for 4 h. After cross-linking, CLEAs-MSG was

ashed for 3 times by phosphate buffer and stored in 0.2 mol/Lhosphate buffer (pH 7.0) at 4 ◦C. The preparing conditions ofLEAs-MSG including buffer pH, enzyme concentration, adsorp-ion time, precipitant type, cross-linker (GA) concentration andross-linking time have all been optimized through single factor

ing Journal 52 (2010) 168–174 169

experiment, i.e. only one factor value was varied to determine theenzyme activity when the other factors were fixed.

2.4. Enzyme activity assay

The activity of free enzyme, CLEAs and CLEAs-MSG were deter-mined by the hydrolysis of casein. In a 10 mL tube, 1 mL of caseinsolution (1%, w/v) in 0.1 mol/L phosphate buffer (pH 7.0) and 1 mLof activation solution (contained 20 mmol cys and 1 mmol EDTA)were added and incubated at 35 ◦C for 10 min. After the incuba-tion, enzyme sample was added. After another 10 min, 2 mL of 10%trichloroacetic acid solution was added into the tube to quench thereaction. The mixed solution was incubated with stirring at 35 ◦Cfor 10 min, and then filtered. The spectrophotometric absorbanceof the supernatant was measured at 275 nm. The hydrolytic activityof enzyme can be calculated as formula (1) described:

D275

Ew × 0.116 × 10= U/mg (1)

where D275 is the absorbance value at 275 nm; Ew is the amount ofenzyme (mg); 0.116 is the absorbance value at 275 nm of 1 �moltyrosine (�mol−1), and 10 is the reaction time (min).

2.5. Optimal conditions for enzyme activity

The optimal temperature of the free enzyme, CLEAs and CLEAs-MSG were determined by adding the enzyme into the substratesolution under different temperatures, and the optimal pH wasdetermined by adding the enzyme into the substrate solution ofdifferent pH. The activities of these immobilized enzyme and freeenzyme samples were determined.

2.6. Storage stability

Storage stabilities of the free enzyme, CLEAs and CLEAs-MSGwere determined by incubating enzyme samples in 0.2 mol/L phos-phate buffer (pH 7.0) without substrate at 4 ◦C. Every 3 days, CLEAsand CLEAs-MSG were separated from the buffer and washed by dis-tilled water. Then the activities of these immobilized enzyme andfree enzyme samples were determined.

2.7. Thermal stability

Thermal stabilities of the free enzyme, CLEAs and CLEAs-MSGwere determined by incubating enzyme samples in 0.2 mol/L phos-phate buffer (pH 7.0) without substrate at 40, 50, 60, 70, and 80 ◦Cfor 10 min. After incubation, the activities of free enzyme, CLEAs andCLEAs-MSG were assayed immediately. Then the residual activitiesat each temperature were determined.

2.8. Recovery stability

The CLEAs-MSG was separated by filter-paper from the reactionsystem after the activity assay, and then washed with 0.2 mol/Lphosphate buffer (pH 7.0) for 3 times to determine the activityagain.

2.9. Kinetically controlled synthesis of z-Ala-Gln

As shown in Fig. 1, z-Ala-Gln was enzymatically synthesized by

kinetically controlled method. 2.193 g (15 mmol) of Gln in 30 mLdistilled water and 0.3536 g (1.5 mmol) of z-Ala-OMe in 1.5 mLethanol were added into a 50 mL flask with stir at 35 ◦C, 160 rpmand adjusted to pH 9.5 with sodium hydroxide. Then the peptidesynthesis reaction was carried out by adding 2400 U free papain

170 M. Wang et al. / Biochemical Engineering Journal 52 (2010) 168–174

etical

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r 2400 U CLEAs-MSG, respectively. The reaction process of pep-ide synthesis and the quantitative analysis of the product wereerformed by HPLC under the following conditions: YMC-PackDS-A C18 column (column i.d. 4.6 mm × 150 mm); isocratic elu-

ion (35% acetonitrile in water with 0.1% trifluoroacetic acid; flowate 1 mL/min; monitor at 214 nm). The yield of z-Ala-Gln was cal-ulated as formula (2):

(%) = cs0 − cs − ch

cs0× 100 (2)

here cs0 and cs were the initial and terminal concentration of z-la-OMe, respectively; and ch was the terminal concentration of-Ala-OH.

All the results in this paper were performed in triplicates.

. Results and discussion

.1. Surface modification of MSG for CLEAs-MSG preparation

In order to stabilize CLEAs in MSG, the silanol groups presentn the porous surface of MSG were modified into amino-groups byPTES because these amino-groups can react with GA which is usu-lly used as a bifunctional agent for enzyme cross-linking. By thisethod, not only enzyme aggregates were cross-linking together to

orm the CLEAs, but also the formed CLEAs were covalently bondedo silica gel at the same time. The IR spectrum measurements wereerformed for the modified MSG. The band assigned to –NH2 was

bserved at 1600 cm−1; the band assigned to N–H was observedt 3400 cm−1; and the band assigned to C–N was observed at100 cm−1. The content of amino-group was 8.5 × 10−3 �mol/mg,hile it could not be determined before modification. These results

ndicated that amino-modified MSG was formed.

Fig. 2. Schematic illustration of the C

ly controlled z-Ala-Gln synthesis.

3.2. Preparation of CLEAs-MSG

The preparation of CLEAs-MSG consisted of three simple stepsas shown in Fig. 2. In the first step, free papains were embeddedin the modified 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 final step, CLEAs were formedand covalently bonded to the MSG through one-step-cross-linking.

3.2.1. Enzyme adsorptionAdsorption is one of the simplest methods for physical immo-

bilization of enzyme since its low cost and mild process [20]. Thismethod benefits from interactions between the carrier surface andthe outer shell of the enzyme. These interactions can be altered bythe pH of the solution, the enzyme concentration and the adsorp-tion time which might affect the adsorption capacity of the carrier[21,22]. Firstly, the effect of pH on the activity in adsorption stepwas studied using papain solutions of different pH values. As shownin Fig. 3a, the highest activity was found to be at pH 6.0–7.0 whichmight be the compromise point of the adsorptive capacity of MSGand the active property of papain. Secondly, in addition to pH value,the activity also depended on the concentration of enzyme solution.Fig. 3b indicated that activity rose with the increase of enzyme con-centration. No significant increasing in activity was observed above0.15 g/mL, which indicated that the enzymes embedded in the MSGwere saturated at this concentration. Thirdly, the effect of adsorp-tion time on activity was shown in Fig. 3c. Before 3 h, more and more

papains diffused into the inner channels and were adsorbed on MSGwith the increasing of time. Thus the amount of embedded enzymeswas the dominant factor for activity. Between 3 and 4 h, the innersurfaces of MSG have been saturated by adsorbed enzymes andthe inner channels have also been saturated by soluble enzymes.

LEAs-MSG preparation process.

M. Wang et al. / Biochemical Engineer

Fig. 3. The effects of (a) enzyme solution pH; (b) enzyme concentration; and (c)adsorption time on the relative activity. Assuming the highest activity value of eachfi37

Tpcmbd

attached to the inner surface of MSG through covalent bonds. Thesebonds were firm enough to avoid CLEAs leaking from the macrop-ores even though the pores were huge to CLEAs. Here, CLEAs-MSGwas prepared under the optimal conditions: 1 g of modified MSGwas incubated in the 0.15 g/mL and pH 7.0 enzyme solution for

gure as 100%. Adsorption conditions: (a) 0.2 g/mL enzyme solution incubated forh at 25 ◦C; (b) pH 7.0 enzyme solution incubated for 3 h at 25 ◦C; (c) 0.2 g/mL, pH.0 enzyme solution incubated at 25 ◦C.

hus the activity of CLEAs-MSG achieved the highest value in thishase. But after 4 h, the autohydrolysis of soluble enzymes in MSG

hannel became serious with the increasing of time [23]. Moreover,ore and more adsorbed enzymes were hydrolyzed by the solu-

le enzymes [24,25]. Thus the autohydrolysis behavior became theominant factor for the rapid decrease of activity in this phase.

ing Journal 52 (2010) 168–174 171

Therefore, the adsorption time was controlled at about 3.5 h toobtain the highest activity.

3.2.2. Enzyme precipitationFree enzymes had been embedded in the MSG after adsorp-

tion. When precipitants were added, enzyme aggregates formedby changing the properties that effect the proximity of the freeenzymes. Usually, the protein recovery agents, such as salts, organicsolvents, non-ionic polymers or acids can be used as precipitants.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 activity was recovered with methanol. 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 fixed.

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-fied 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 of CLEAs-MSG. These resulted from the property of GAwhich was not only a cross-linking agent but also a denaturantfor enzyme. The active conformation might be ruined if the cross-linking reaction affected the active sites of enzyme. The high GAconcentration and the long reaction time might intensify the lossof enzyme activity. Moreover, at the high GA concentration theundesired excessive self-cross-linking among enzyme aggregatesand among MSG surfaces would compete the effective sites for thedesired one-step-cross-linking between CLEAs and MSG surfaces.Therefore, we controlled the GA concentration to be 2% and limitedthe reaction time to be 4 h in order to obtain the highest activ-ity of CLEAs-MSG.In a word, by one-step-cross-linking, CLEAs were

Fig. 4. The effect of precipitants on the papain activity. Assuming the activity with-out precipitation as 100%. Precipitation conditions: 4 mL precipitant (saturatedammonium sulfate, anhydrous alcohol, 100% acetone or 100% methanol) per gramenzyme-adsorbed MSG, pH 7.0 and 25 ◦C.

172 M. Wang et al. / Biochemical Engineering Journal 52 (2010) 168–174

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free enzyme. However, CLEAs-MSG did not increase the opti-

F

ig. 5. The effects of cross-linking time and GA concentration on the activity ofLEAs-MSG. Assuming the CLEAs-MSG activity value under 2% GA concentrationnd 4 h cross-linking as 100%.

.5 h at 25 ◦C, and then precipitated with 4 mL anhydrous alcoholnd cross-linked with 2% GA for 4 h. The resulting activities were60 U/g CLEAs-MSG and 14,000 U/g CLEAs.

.3. Structural characterization by SEM

The SEM image of modified MSG before immobilization washown in Fig. 6a and c, and the CLEAs-MSG was shown in Fig. 6bnd d, respectively. It could be observed in Fig. 6a (magnificationf 5000×) that the pores and channels of modified MSG were largenough for free enzymes to pass through without much diffusionimitation and for CLEAs to be accommodated. As shown in Fig. 6bmagnification of 5000×), CLEAs were formed in these macrop-res as CLEAs-MSG, and the CLEAs entrapped in the pores would

ot leak out because the CLEAs were covalently bonded to MSGhrough one-step-cross-linking. This attachment could be obvi-usly observed in Fig. 6c and d (magnification of 20,000×) thathe surface of the MSG was very smooth (Fig. 6c) but became

ig. 6. SEM images of (a) modified MSG magnified 5000×; (b) CLEAs-MSG magnified 500

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 filtration because of the supporting effect of MSG.

3.4. The optimal biocatalytic conditions of free enzyme, CLEAsand CLEAs-MSG

3.4.1. The optimal temperatureThe optimal temperature was at 80 ◦C for free papain, 50–80 ◦C

for CLEAs and 40–90 ◦C for CLEAs-MSG as shown in Fig. 7. Com-monly, immobilization can increase the optimal temperature of

mal temperature obviously but extended the optimal temperaturerange. It could be seen that CLEAs-MSG maintained the high activityin a wider temperature range compared to free papain and CLEAs.This improvement resulted from the covalent cross-linking of GA

0×; (c) modified MSG magnified 20,000×; (d) CLEAs-MSG magnified 20,000×.

M. Wang et al. / Biochemical Engineering Journal 52 (2010) 168–174 173

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ig. 8. The optimal pH curves of free and immobilized enzyme. Assuming the highestctivity of free enzyme, CLEAs and CLEAs-MSG as 100%, respectively.

mong enzymes and between CLEAs and MSG surfaces. This struc-ure stabilized the conformation of active sites of enzyme, whichould be easily ruined by the change of reaction temperature.

.4.2. The optimal pHAs shown in Fig. 8, the optimal pH values of free papain and

LEAs-MSG were observed as 7.0, but that of CLEAs was as 6.0.his shift might be caused by the ionization changes of acidic orasic amino acid side chains in the microenvironment around thective site of enzyme or on the surface group of MSG [26]. Besides,LEAs-MSG retained the higher activity above pH 7.0 and belowH 4.0 compared with free enzyme and CLEAs, which indicatedhat CLEAs-MSG protected papain from intensively deactivating atarsh conditions.

.5. The stability of free enzyme, CLEAs and CLEAs-MSG

.5.1. Storage stability

To investigate the effect of immobilization on the storage sta-

ility of papain, free enzyme, CLEAs and CLEAs-MSG were stored at◦C in aqueous buffer. As shown in Fig. 9, free enzyme lost almost

ts total activity after only 3 days, but CLEAs and CLEAs-MSG bothetained about 50% of their initial activities after 3 days and about

ig. 9. The storage stability of free and immobilized enzyme in aqueous buffer.ssuming the initial enzyme activity before incubation as 100%.

Fig. 10. The thermal stability of free and immobilized enzymes at different temper-atures. Assuming the initial enzyme activity (determined at 35 ◦C) before incubationas 100%. Incubation conditions: pH 7.0 aqueous buffer, incubated for 10 min.

35% after 15 days. These results demonstrated that CLEAs-MSG hadno significant enhancement on the storage stability comparing withthat of CLEAs but dramatically improved the store stability of freepapain. It is because that the multipoint immobilization of CLEAs-MSG and CLEAs prevent the dissociation of enzyme from its matrix(MSG or enzyme aggregates) and further prevent the leaking ofenzyme into aqueous buffer.

3.5.2. Thermal stabilityThe thermal stability of free and immobilized papain were

determined by incubating them in aqueous buffer (pH 7.0) at dif-ferent temperatures for 10 min. The residual activities were givenin Fig. 10. It could be seen that the CLEAs-MSG retained more than83% of its initial activity at each temperature after 10 min of incu-bation which was a little higher than that of CLEAs (residual activitywas about 70–80%), but the free papain lost its initial activity dra-matically (residual activity was about 20–50%) after incubation.These results indicated that the thermal stability of CLEAs-MSG wasmuch better than that of free enzyme and CLEAs. This enhancementof CLEAs-MSG in thermal stability might be due to the covalentinter-cross-linking between CLEAs and MSG surfaces as well asthe covalent intra-cross-linking among enzyme aggregates. Due tothese covalent bonds, CLEAs-MSG needed much more energy tobreak down the active conformation than free enzyme and CLEAs.In another word, it was more difficult to ruin the conformation ofenzyme in CLEAs-MSG than that of free enzyme and CLEAs if theywere under the same thermal condition.

3.6. The recovery of CLEAs-MSG

As mentioned above, the small size and low compressive resis-tance of CLEAs made it difficult to recover and apply to someindustrial fields. However, CLEAs-MSG overcame these problems.The CLEAs-MSG pellets were of 5 mm diameter as we prepared, andthe size could be also adjusted according to the practical needs.The proper size of CLEAs-MSG facilitated the separation of bio-catalyst from reaction system only by filtration. The initial andrecovered activities of CLEAs-MSG for each determination were

100%, 97.3%, 89.8% and 76.1%. That meant CLEAs-MSG still retainedthe high activity after using for 4 times. These results indicatedthat CLEAs-MSG improved the properties of CLEAs in practicalapplication.

174 M. Wang et al. / Biochemical Engineer

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ig. 11. The time course in kinetically controlled synthesis of z-Ala-Gln. Reactiononditions: 0.5 mol/L z-Ala-OMe, 0.05 mol/L Gln, 35 ◦C, pH 9.5, 160 rpm and 2400 Unzyme.

.7. The application of CLEAs-MSG to kinetically controlledynthesis of z-Ala-Gln

Since the excellent stability and recovery, CLEAs-MSG waspplied in kinetically controlled synthesis of z-Ala-Gln. As shownn Fig. 11, the time course in synthetic performance of z-Ala-Glny CLEAs-MSG was quite similar to that by free enzymes when thequal units of activity (2400 U) were added. The final yield was2.9% at 140 min by using CLEAs-MSG as biocatalyst and 31.9% bysing free enzyme. This result indicated that CLEAs-MSG not onlyad the equivalently catalytic ability to free enzyme in kineticallyontrolled synthesis of z-Ala-Gln, but also do not change the reac-ion equilibrium and the selectivity of papain. In addition, the hightability of CLEAs-MSG in aquatic solvents facilitated the isolationnd purification of reaction products.

. Conclusions

A simple method for preparing CLEAs of papain in the macro-ores of silica gel (CLEAs-MSG) was proposed. The inner channelsf MSG were large enough to accommodate CLEAs and preventedLEAs from leaking out effectively by the one-step-cross-linkingtrategy. It was demonstrated that pH of enzyme solution, enzymeoncentration, adsorption time, precipitant type, GA concentra-ion and cross-linking time had significant effects on CLEAs-MSGctivity in the process of preparing CLEAs-MSG. The CLEAs-MSGf papain displayed the improved optimal temperature and pHonditions, and exhibited the higher storage stability and thermaltability than free papain and in some cases CLEAs. Additionally,imilar to free papain, CLEAs-MSG catalyzed the synthesis of z-Ala-ln with the yield of 32.9% through kinetically controlled method.he convenient preparing process and the excellent propertiesf CLEAs-MSG reported in this paper may provide a feasible andfficient solution to the drawbacks of enzymes in industrial appli-ation.

cknowledgements

The authors thank the financial support from the Program forew Century Excellent Talents in Chinese University (NCET-08-386), the Key Project of Chinese Ministry of Education (108031),

[

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ing Journal 52 (2010) 168–174

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).

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