crosslinked enzyme aggregates in hierarchically-ordered mesoporous silica: a simple and effective...

ARTICLE

Crosslinked Enzyme Aggregates inHierarchically-Ordered Mesoporous Silica:A Simple and Effective Methodfor Enzyme Stabilization

Moon Il Kim,1 Jungbae Kim,2 Jinwoo Lee,3 Hongfei Jia,4 Hyon Bin Na,3 Jong Kyu Youn,1

Ja Hun Kwak,2 Alice Dohnalkova,2 Jay W. Grate,2 Ping Wang,4 Taeghwan Hyeon,3

Hyun Gyu Park,1 Ho Nam Chang1

1Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of

Science and Technology (KAIST), Daejeon 305-701, Republic of Korea;

telephone: þ82-42-869-3932; fax: þ82-42-869-3910; e-mail: [email protected] Northwest National Laboratory, Richland, Washington 993523National Creative Research Initiative Center for Oxide Nanocrystalline Materials and

School of Chemical Engineering, Seoul National University, Seoul, Republic of Korea4Department of Chemical Engineering, University of Akron, Akron, Ohio

Received 12 April 2006; accepted 29 June 2006

Published online 19 September 2006 in Wiley InterScience (www.interscience.wiley.c
om). DOI 10.1002/bit.21107
ABSTRACT: a-chymotrypsin (CT) and lipase (LP) wereimmobilized in hierarchically-ordered mesocellular meso-porous silica (HMMS) in a simple but effective way forthe enzyme stabilization, which was achieved by theenzyme adsorption followed by glutaraldehyde (GA) cross-linking. This resulted in the formation of nanometer scalecrosslinked enzyme aggregates (CLEAs) entrapped in themesocellular pores of HMMS (37 nm), which did notleach out of HMMS through narrow mesoporous channels(13 nm). CLEA of a-chymotrypsin (CLEA-CT) in HMMSshowed a high enzyme loading capacity and significantlyincreased enzyme stability. No activity decrease of CLEA-CTwas observed for 2 weeks under even rigorously shakingcondition, while adsorbed CT in HMMS and free CTshowed a rapid inactivation due to the enzyme leachingand presumably autolysis, respectively. With the CLEA-CTin HMMS, however, there was no tryptic digestion observedsuggesting that the CLEA-CT is not susceptible to autolysis.Moreover, CLEA of lipase (CLEA-LP) inHMMS retained 30%specific activity of free lipase with greatly enhanced stability.This work demonstrates that HMMS can be efficientlyemployed as host materials for enzyme immobilizationleading to highly enhanced stability of the immobilizedenzymes with high enzyme loading and activity.

Biotechnol. Bioeng. 2007;96; 210–218.

� 2006 Wiley Periodicals, Inc.

Correspondence to: H.G. Park

Contract grant sponsor: Basic Research Program of the Korea Science & Engineering

Foundation

Contract grant number: R01-2004-000-10293-0

Contract grant sponsor: Brain Korea 21 project of the Ministry of Education

This article contains Supplementary Material available via the Internet at http://

www.interscience.wiley.com/jpages/0006-3592/suppmat.

210 Biotechnology and Bioengineering, Vol. 96, No. 2, February 1, 2007

KEYWORDS: CLEAs (crosslinked enzyme aggregates);a-chymotrypsin; Mucor javanicus lipase; enzyme immobi-lization; HMMS (hierarchically-ordered mesocellularmesoporous silica)

Introduction

Enzymes are highly selective and efficient catalysts that areincreasingly attractive to many important chemical proces-sing applications. The specificity of enzymes promises greatadvancements in various applications such as chemicalconversions, biosensing, and bioremediation (Duran andEsposito, 2000; Koeller and Wong, 2001; Schmid et al.,2001). However, their widespread utilization is often limitedby a low operational stability. Therefore, there have beenmany studies to stabilize enzyme activity including enzymeimmobilization, enzyme modification, genetic modifica-tion, and medium engineering (DeSantis and Jones, 1999;Kim and Grate, 2003; Livage et al., 2001; Tischer andWedekind, 1999).

Of these stabilization methods, immobilization strategyusing solid supports has been most extensively researched

� 2006 Wiley Periodicals, Inc.

because the immobilized enzymes have several specificadvantages. First, the immobilized enzymes are easilyrecovered from the reaction medium for their reuse.Second, the immobilization of enzymes in a solid matrixlimits their conformational variations diminishing unpre-dictable changes of their characteristic properties, whichoften occurs in enzyme modification. Moreover, the solidmatrix may serve as a shield for harsh environmentalconditions like pH variation, temperature alteration, andshaking condition.

Immobilization method using crosslinking reactionbetween enzyme molecules is an attractive strategy becauseit affords stable catalysts with high retention of activity (Caoet al., 2000; Schoevaart et al., 2004). The multipointattachment of enzyme molecules is well known to stabilizethe enzyme activity by effectively preventing enzymedenaturation (Mozhaev et al., 1990). By crosslinking reactiveamine residues of the enzyme with glutaraldehyde (GA),crosslinked enzymes (CLEs) (Doscher and Richards, 1963)and crosslinked enzyme crystals (CLECs) (Haring andSchreier, 1999) were developed to increase the operationalstability of enzymes and to facilitate their recovery andrecycling. Recently, Cao et al. (2000) developed such anapproach and named the resulting enzymes as crosslinkedenzyme aggregates (CLEAs). The procedure for makingCLEAs includes physical precipitation followed by GAcrosslinking. The resulting CLEAs proved to be active andrecyclable using tens of enzymes (Schoevaart et al., 2004).However, CLEAs are mechanically fragile, and it is difficultto handle and fully recover the CLEA particles overrepetitive uses (Khare et al., 1991; Wilson et al., 2004).

Khare et al. (1991) tried to develop an enzyme aggregatesystem by crosslinking enzyme molecules inside Sephadexbeads, but the final enzyme loading and stability were notimpressive even in a static non-shaking condition. Poly-meric beads do not seem to be a good host material for suchan entrapment approach because their pore size cannot bewell controlled for desirable defined structure and they aresusceptible to structural deformation, which consequentlyleads to a serious enzyme leaching and poor enzyme loading.

Recently, inorganic mesoporous silica materials haveattracted much attention as a host of enzymes because oftheir controlled porosity and high surface area (Davis, 2002;Han et al., 1999; Lee et al., 2001; Lei et al., 2002; Schmidt-Winkel et al., 1999; Ying et al., 1999). A simple adsorptionmethod has been most frequently adopted to immobilizeenzymes into mesoporous materials (Dıaz and Balkus, 1996;Fan et al., 2003a, 2003b; Han et al., 1999, 2002; Lei et al.,2002; Takahashi et al., 2000). This approach, however,would not maintain the initially loaded enzymes in acontinuous operation due to the leaching of enzymes. Toprevent the leaching problem, enzyme molecules can beattached to mesoporous materials via covalent linkage(Chong and Zhao, 2004; Kim et al., 2004; Wang et al., 2001).However, the final loading of enzymes is still poor due totwo reasons: only the inner surface of mesopores (ratherthan the whole pore volume) is used for the attachment of

enzymes; and the attached enzyme molecules can exert aserious steric hindrance against the penetration of otherenzyme molecules into deeper mesopores.

Very recently, we developed a simple strategy for thesynthesis of hierarchically-ordered mesocellular mesopor-ous silica (HMMS) materials having uniform porestructures with two different length scales, and brieflydemonstrated their successful application as a host ofenzyme immobilization (Kim et al., 2005; Lee et al., 2005).To intensively verify the applicability of the novel materialsin enzyme immobilization, we describe herein, a simple andeffective strategy for enzyme stabilization using CLEAs inHMMS. Using GA as a crosslinking agent, we preparednanometer-scale CLEAs of a-chymotrypsin (CT) and lipase(LP) in HMMS. CT and LP were employed as modelenzymes due to their widespread use in various industrialareas including preparation of peptide and chiral molecules(Gandhi et al., 2000; Santaniello et al., 1988). The effects ofGA concentration and initial enzyme concentration on theenzyme loading, activity, and stability were investigated.Particularly, the stability of CLEAs was measured in arigorously shaking condition. There has been little reportpublished about the evaluation of enzyme stability undersuch a harsh condition due to a serious enzyme leachingproblem, which was successfully resolved by using thepresent strategy.

Materials and Methods

Materials

a-chymotrypsin (CT), glutaraldehyde (GA), N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (TP), 4-nitrophenyl buty-rate were purchased from Sigma Chemical (St. Louis, MO).Mucor javanicus lipase was from Fluka (Milwaukee, WI). Allother reagents and solvents were purchased from AldrichChemical (Milwaukee, WI) and were of the highest gradecommercially available.

Synthesis and Characterization of HMMS(Mesocellular Mesoporous Silica)

The synthesis of HMMS was done by following a protocol ofLee et al. (2005). Typical synthesis procedure for HMMS isas follows: 9.7 g of P123 ((EO)20(PO)70(EO)20) and 4.48 mLacetic acid was dissolved in 200 mL water. The solution washeated to 608C andmaintained at that temperature for morethan 1 h. Sixteen milliliters sodium silicate (27% SiO2, 14%NaOH) diluted with 200 mL of water was poured into theprepared solution with vigorous stirring. The pH of thesynthetic solution was 6.3� 6.4. The solution was furtherheated to 608C and aged at that temperature for 20 h,followed by hydrothermal treatment at 1008C for 24 h.Calcination of filtered materials at 5508C generated HMMS.

Kim et al.: Crosslinked Enzyme Aggregates in Mesoporous Silica 211

Biotechnology and Bioengineering. DOI 10.1002/bit

The HMMS particles were suspended in an acrylic resin,and cured in 608C overnight. The polymerized blocks weresectioned on a Leica UCT ultramicrotome to a thickness of20 nm. The sectioned samples were examined in JEOL 2010TEM operating at 200 kV. FE-SEM images were taken by aJEOL JSM-6700F instrument. Nitrogen adsorption mea-surement was carried out using a Micrometrics model ASAP2010 adsorption analyzer. Before analysis HMMS samplewas degassed for more than 4 h at 2008C under vacuum inthe degas port.

Enzyme Immobilization in HMMS

HMMS (10 mg) was mixed with 1.5 mL of free CT in abuffer solution (10 mM sodium phosphate buffer, pH 7.8),vortexed for 30 s, sonicated for 3 s, and incubated at roomtemperature in a shaking condition (250 rpm). Variousconcentrations of free CT (0.5, 1, 2, and 4 g/L) were used forthe enzyme adsorption in HMMS. After 20-min incubationfor adsorption of free CT in HMMS, the samples werewashed briefly by an aqueous buffer (100 mM sodiumphosphate buffer, pH 8.0), and incubated in the same buffercontaining various GA concentrations (0, 0.01, 0.05, 0.1, and1% w/w) at 200 rpm for 30 min. After GA treatment, thesamples were washed by a phosphate buffer (100 mMsodium phosphate, pH 8.0) and 100 mM Tris-HCl buffer(pH 8.0). The capping of unreacted aldehyde groups wasperformed in a fresh Tris-HCl buffer (100 mM Tris, pH 8.0)at 200 rpm for 30 min. After Tris-capping, the samples werewashed twice by phosphate buffer (10 mM sodiumphosphate buffer, pH 7.8), and stored at 48C. The proteinleaching amount from HMMS into the supernatant wasmeasured by BCA (PIERCE, Rockford, IL) and Lowry (Bio-Rad, Hercules, CA) method (Smith et al., 1985) at eachwashing step. The final enzyme loading in HMMS wascalculated from difference between the initial CT amountand the leached amount of CT into washing solutions.CLEA-LP was prepared by the same experimental proce-dure, with 1.2 g/L initial enzyme concentration and 0.1%(w/w) GA concentration.

Enzyme Activity

The activities of free CT, immobilized CT, and CLEA-CTwere measured by the hydrolysis of N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (TP) in an aqueous buffer (10 mMsodium phosphate buffer, pH 7.8) at room temperature. Theabsorbance increase at 410 nm was monitored using aspectrophotometer (Model Cary 5G UV-Vis-NIR spectro-photometer from Varian, Inc., Palo Alto, CA), andconverted to the initial hydrolytic rate of TP at each TPconcentration. For the activity measurement of immobilizedenzyme systems, a small aliquot of samples was mixed withTP (10� 200 mM) and shaken at 250 rpm at roomtemperature. The increase of absorbance at 410 nm in thesupernatant was measured time-dependently after centri-


fugation at 5,000g. The active site concentrations weredetermined by the MUTMAC assay (Gabel, 1974).

The activities of free LP, CLEA-LP, and adsorbed-LP weremeasured by the hydrolysis of 4-nitrophenyl butyrate in anaqueous buffer (10mM sodium phosphate buffer, pH 6.5) atroom temperature. The absorbance increase at 400 nm wasmonitored using the spectrophotometer (Model Cary 5GUV-Vis-NIR spectrophotometer from Varian, Inc.). For theactivity measurement of immobilized enzyme systems, asmall aliquot of samples was mixed with 4-nitrophenylbutyrate (500 mM) and shaken at 250 rpm at roomtemperature. The increase of absorbance at 400 nm in thesupernatant was measured time-dependently after centri-fugation at 5000g.

Enzyme Stability

The enzyme stability was checked in aqueous buffer (10 mMsodium phosphate, pH 7.8 for CT and pH 6.5 for LP) in ashaking condition (200 rpm). After each time point, theresidual activity of each sample was determined bymeasuring hydrolytic enzyme activity of substrates asdescribed above. The relative activity (%) was calculatedby the ratio of the residual activity to the initial activity ofeach sample. The resistance to tryptic digestion was alsochecked by measuring the residual activity after incubationof each sample in the presence of trypsin (TR).

Large-Sized CLEA-CT Without HMMS

Fifty milligrams CT was dissolved in 1 mL buffer (100 mMsodium phosphate, pH 7.8) in a 15 mL centrifuge tube. Onemilliliter of a 55% (w/v) ammonium sulfate solution in abuffer, and 83 mL GA (25% in water) were added. Themixture was stirred at 48C for 17 h. Five milliliters of freshsodium phosphate buffer (10 mM, pH 7.8) was added andthe mixture was centrifuged. After the supernatant wasdecanted, the sample was washed with the buffer (5 mL eachtime) four times with a cycle of centrifugation anddecantation. The final CLEAs (called large-sized CLEAs)was kept at 48C in 5 mL of sodium phosphate buffer(10 mM, pH 7.8).

Results and Discussion

Preparation of CLEAs-CT in HMMS

The preparation of CLEAs in HMMS consists of two simplesteps as shown in Scheme 1. The first step is to adsorbenzymes in HMMS and the second step is to crosslinkthe adsorbed enzymes via GA treatment, resulting innanometer-scale CLEAs in the mesopores of HMMS. Thestructural properties of the employed HMMS were shown inFigure 1. SEM image of the HMMS (Fig. 1a) indicates thatthe component size of HMMS ranges from 200 to 500 nm.

DOI 10.1002/bit

Scheme 1. Schematic representation for CLEAs in HMMS (crosslinked enzyme aggregates in hierarchically ordered mesocellular mesoporous silica). [Color scheme can be

seen in the online version of this article, available at http://www.interscience.wiley.com.]

TEM image of the HMMS (Fig. 1b) shows the co-existenceof large mesocellular pores (37 nm) and small mesoporouschannels (13 nm). The channels are large enough for theenzymes to pass through without considerable diffusionlimitation and the mesocellular pores can accommodateCLEAs. We expect that the CLEAs entrapped in themesocellular pores (37 nm) would not leach out throughthe mesoporous channels (13 nm), like in a ship-in-a-bottleapproach, leading to high enzyme loading with concomitantimproved stabilization of enzyme activity.

Under shaking condition (250 rpm) in a slanted positionat room temperature (228C), CT adsorption was completedin less than 5 min with a loading of up to 570 mg/g (36.3wt%) when HMMS was incubated in 4 g/L CT solution.Since the pore volume of HMMS is 1.34 mL/g, thetheoretical maximal CT loading value in the HMMS wasapproximately 904 mg/g (47.5 wt%, see SupplementaryInformation), indicating that the enzyme loading is 63% ofthe theoretical maximum value. The unloaded 37% spacecan be explained by the existence of smaller pores than thesize of CT molecule and imperfect packing. Despite the highinitial adsorption of CT, the physically adsorbed CTseriously leached out of HMMS. In a typical experiment,nearly half of the adsorbed CT leached out into the washingsolutions during the first five washing steps, with eachwashing performed under shaking (200 rpm) for 20 min.

To prevent the leaching problem, adsorbed CT wastreated with various GA concentrations (0, 0.01, 0.05, 0.1,and 1% w/w). With this treatment, the loading capacity ofthe resulting CT was significantly improved even after five-time washing steps (Table I). The improved loading capacityranged from 30.3 to 33.2 wt%, and there was no bigdifference depending on the used GA concentration in the

tested range from 0.01 to 1%, indicating that 0.01% GAconcentration is effective enough to prevent the CTmolecules from leaching out. This improvement of CTloading via GA treatment supports our initial hypothesis inScheme 1, which represents a ship-in-a-bottle approachbased on the combination of enzyme crosslinking andunique pore structure of HMMS. Without GA crosslinking(GA 0%), adsorbed CT in HMMS continuously leached outfrom HMMS during washing, and the final loading waslowered to 21.0 wt% from the initial 36.3 wt%.

Stability of CLEAs-CT in HMMS

Prevention of CT leaching is supposed to be accompanied bythe improved enzyme stability and these effects wereinvestigated. The stabilities of free CT, adsorbed CT, andCLEAs-CT in HMMS were evaluated by incubating them inaqueous buffer under shaking (200 rpm) in a horizontalposition (side-by-side). Up to now, little report hasemployed this rigorously shaking condition for the stabilitytest. Most of the researchers just incubated the samples in astatic non-shaking condition for the evaluation of thestability of immobilized enzymes in mesoporous silica (Dıazand Balkus, 1996; Fan et al., 2003a, 2003b; Han et al., 1999,2002; Takahashi et al., 2000). Commercial enzymaticprocess, however, are usually conducted under such a harshshaking condition to relieve the mass transfer limitation ofsubstrate and product in the heterogeneous catalyst system,and therefore the immobilized enzyme should be stabilizedunder shaking condition for its successful application.

Figure 2 shows the stability of CLEAs-CT treated withvarious concentrations of GA after adsorption of CT (4 g/L



Figure 1. a: SEM image of HMMS component. b: TEM image of HMMS. Co-

existence of large mesocellular pores and small mesoporous channels can be shown.

Figure 2. Stability of CLEAs-CT_HMMS prepared with 4 g/L CT and various

glutaraldehyde concentration at 228C. The relative activity (%) represents the ratio of

residual activity to initial activity of each sample. [Color figure can be seen in the online

version of this article, available at http://www.interscience.wiley.com.]

CT solution) into HMMS. The relative activity, defined asthe percentage of residual activity to the initial activity ofeach sample, was calculated at each time point. Treatmentswith relatively low GA concentrations (0.01 and 0.05% GA)resulted in a marginal improvement of enzyme stability and

Table I. Loading capacity and activity of CLEAs-CT and adsorbed-CT in HM

Samplesb Adsorbed-CT

C

(G

CT loading amount in HMMS (wt%) 21.0

Specific activity (mmol/min per mg CT) 0.91

Specific activity (mmol/min per mg silica) 0.26

% of active site per protein contentd 19%

aThe experiments were repeated five times and the results were averaged. CbAll immobilized samples were made using 4 g/L CT solution.cX in the sample names (GA X%) represents the GA concentration used fodThe active site content was determined by the MUTMAC assay (Gabel, 19


there was still CT leaching observed with the samples, whichwas confirmed by the measurement of the leached CT in thesupernatant. Upon increasing the GA concentrations to 0.1or 1%, the CT stability greatly improved. With thesesamples, there was no measurable decrease in CT activitydetected for up to 2-week incubation period under shaking.Two key factors are considered to be responsible for thisimpressive stability of CLEA-CT. The first one is theprevention of CT leaching in a specially designed HMMS aspreviously described and the second one is the inhibition ofautolysis by GA crosslinking (Migneault et al., 2004; Roy andAbraham, 2004). The control experiment without GAtreatment (GA 0%) showed a continuous loss of CT activity.

To investigate the ship-in-a-bottle nature of CLEA inHMMS, more diverse CLEAs-CT were prepared by varyingthe concentration of CT (0.5, 1, 2, and 4 g/L) for initialadsorption. All the samples were treated with 0.1% GAsolution because 0.1% appeared to be effective enough forthe stability improvement based on the previous experiment(Fig. 2). Control samples of adsorbed CT were also preparedby using the same CT concentration and same procedure,but without GA treatment. The results for the CT loadings of

MS prepared with various GA concentrationsa.

LEA-CT

A 0.01%)cCLEA-CT

(GA 0.05%)cCLEA-CT

(GA 0.1%)cCLEA-CT

(GA 1%)c

30.3 32.6 33.2 32.0

1.29 1.09 0.88 0.47

0.56 0.53 0.44 0.22

29% 25% 23% 27%

oefficients of variation (CV) of the results were less than 7%.

r crosslinking.74).

DOI 10.1002/bit

Table II. CT loading of CLEAs-CT and adsorbed-CT in HMMSa.

Samplesb

CT

loading in

HMMS (%)

Estimated number

of enzyme

molecules in each

mesocellular porec

Maximal CT loadingc 47.5 499

CLEA-CT (GA 0.1%) (4 g/L CT) 33.2 274

CLEA-CT (GA 0.1%) (2 g/L CT) 19.7 135

CLEA-CT (GA 0.1%) (1 g/L CT) 10.8 67

CLEA-CT (GA 0.1%) (0.5 g/L CT) 6.0 35

Adsorbed-CT (GA 0%) (4 g/L CT) 21.0 —



Adsorbed-CT (GA 0%) (0.5 g/L CT) 4.6 —

aAll experiments were repeated three times and the results were averaged.Coefficients of variation (CV) of the results were less than 7%.

bX in the sample name (X g/L CT) represents the CT concentration forthe enzyme adsorption. All samples were washed five times, and eachwashing was done under rigorous shaking (200 rpm) for 20 min.

cSee Supplementary Information for detailed calculation.

CLEA-CT and adsorbed CT are summarized in Table II. Inboth cases, more CT loadings were obtained with higher CTconcentration used for initial adsorption, and the CTloading of CLEA-CT was always higher than that ofcorresponding adsorbed CT, as expected. With 4 g/L of CTconcentration, relatively higher improvement of the CTloading was caused by the GA treatment when comparedwith lower CT concentrations. This suggests that highlypopulated CT molecules in the mesopores of HMMS lead tomore rigorous formation of CT aggregates, which canprevent the enzyme leaching more efficiently.

The stabilities of CLEAs-CT with different CT loadingswere compared to those of free CT and adsorbed CT withoutGA treatment (Fig. 3). There was a rapid decrease observedwith the activity of free CT and less than 1% of initial activity

Figure 3. a: Stability of CLEAs-CT_HMMS, adsorbed-CT_HMMS, and Free-CT in a sha

and 0.1% glutaraldehyde concentration. The concentration in the sample name represen

CT_HMMS in a linear scale representation of Y-axis. [Color figure can be seen in the on

remained after 8 h incubation. This must be due to thecombined effect of rapid denaturation and autolysis undershaking (Hofstee, 1965). Adsorption of the enzyme intoHMMS improved the CT stability to some extent whencompared to free CT, and the residual activities of adsorbedCT were 24–50% of initial activities after 9 h incubation.However, after 6-day incubation, the residual activities werereduced to only 1–2% of initial activities, regardless of theCT concentrations for adsorption (Fig. 3a). This activityreduction was due to the leaching of CT fromHMMS, whichwas confirmed by the measurements of leached CT in thesolution.

In the case of CLEAs-CT, relative activities were greatlyimproved for all different CT concentrations due to the GAcrosslinking. With the samples prepared by 2 and 4 g/L CT,there was no measurable decrease in their relative activitieswhile the samples from lower CT concentrations (0.5 and1 g/L CT) showed about 22–23% decrease of their activityafter 6-day incubation (Fig. 3b). This decrease of CT activitywith lower CT concentrations emphasizes the importance ofhighly populated CT molecules during GA treatment for thesuccess in preventing the CT leaching and stabilizing the CTactivity. As a possible explanation, the size of CT aggregateswould become smaller when lower CT concentrations wereused for adsorption, as can be presumed from the loweredCT loadings in HMMS consequently leading to the smallernumber of estimated CT molecules in each mesocellularpore of HMMS (Table II). The smaller CT aggregates wouldmore easily leach out of HMMS through mesoporouschannels (13 nm). On the other hand, higher CTconcentrations would lead to highly populated CTmolecules in the mesopores of HMMS right after adsorp-tion, and GA crosslinking would result in larger CTaggregates that cannot leach out of HMMS through smallermesoporous channels.

king condition (200 rpm) at 308C. CLEAs were prepared with various CT concentrationts the initial CT concentration for the adsorption into HMMS. b: Stability of CLEAs-

line version of this article, available at http://www.interscience.wiley.com.]



Figure 4. a: Schematic representation for the inhibition of proteolytic digestion of CLEAs in HMMS (crosslinked enzyme aggregates in mesocellular mesoporous silica).

b: Proteolysis measurement of CLEA-CT_HMMS, adsorbed-CT_HMMS, large-sized CLEA-CT, and their mixtures with trypsin in a vertically shaking condition (200 rpm) at 228C. CTsolution of 4 g/L was used for enzyme adsorption and 0.1% glutaraldehyde was used for CT crosslinking in HMMS. Large-sized CLEA-CT was prepared by the procedure described in

Materials section. The relative activity (%) represents the ratio of residual activity to initial activity of each sample. [Color figure can be seen in the online version of this article,

available at http://www.interscience.wiley.com.]

Since the impressive stability of CLEA-CT in HMMSsuggests the effective inhibition of CT autolysis, we alsoinvestigated the resistance of CLEAs against proteolyticdigestion (Fig. 4). The proteolysis of CLEA-CT wasperformed by incubating the samples with anotherproteolytic enzyme, TR. The proteolytic activity wasdetermined by measuring the residual CT activity becausethe proteolysis would decrease the CT activity (Hedstrom,1996). Since TR showed negligible activity toward thesubstrate of CT, N-Succinyl-Ala-Ala-Pro-Phe p-nitroani-lide, there was no interference of TR for the measurement ofCT activity. Adsorbed-CT in HMMS and large-sized CLEA-CT without HMMS were also included in this experimentfor comparison. Without TR, the activity of CLEAs-CT inHMMS and large-sized CLEA-CT did not decrease at allduring 40 h incubation at 200 rpm shaking conditionwhereas adsorbed-CT showed a continuous decrease of CTactivity. Upon the addition of TR, the activity of large-sizedCLEA-CT significantly decreased to only 60% of its initialactivity in 40 h incubation due to the tryptic digestion. Onthe other hands, CLEAs-CT in HMMS did not show anyactivity decrease even with the addition of TR. This can beascribed to the fact that the HMMS matrix protected theimmobilized CT from the tryptic digestion, providing anadditional shielding effect. In the case of the adsorbed-CT inHMMS, the addition of TR caused a further activity loss,indicating that the shielding effect of HMMS is not goodenough and the GA crosslinking is essential for completeprevention of autolysis and proteolysis.


Activity of CLEA-CT in HMMS

The activities of immobilized CT in HMMS were alsoinvestigated. Upon increasing GA concentration, the activityof the CLEAs-CT decreased (Table I) and this trend isconsistent with the notion that the higher GA concentrationwould cause the more structural perturbation of the CTmolecules (DeSantis and Jones, 1999). Considering theprevious results that higher concentration of GA is requiredfor a good stability of the enzyme, there should be acompromising point for an optimal GA concentration.Based on the present experimental results, the 0.1% GAtreatment appears to be best, providing a high loading (33.2wt%), improved stability, and good activity. GA 1%concentration significantly decreased the activity withoutfurther improvement of the loading capacity (32.0 wt%) orstability, while measurable enzyme leaching was observedwith lower GA concentrations as previously mentioned.

Even with the high loading and long-term stability ofCLEA-CT, its specific activities were about 20 times lowerthan that of free CT (26.9 mmol/min per mg CT). Wesuspected that the lowered activity of CLEA-CT may be dueto the autolysis of CT molecules during the immobilizationprocess (Kim et al., 2005). To check the autolysis during theenzyme immobilization, we measured the number ofactive sites in each sample, and calculated the ratio of theactive site number to the total number of loaded enzymemolecules (Table I). Interestingly, this ratio was reducedfrom 61� 2% of free CT to 26� 3% and 19� 3% of

DOI 10.1002/bit

Table III. Enzyme loading and activity of CLEA-LP, adsorbed-LP in HMMS, and free-LPa.

Samplesb CLEA-LP Adsorbed-LP Free-LP

LP loading amount in HMMS (%) 16.3 13.0 —

Specific activity (mmol/min per mg LP) 0.23 0.27 0.78

Specific activity ratio (%) 29.8 34.1 100.0

Activity per unit HMMS (mmol/min per mg MMS) 0.045 0.040 —

aThe experiments were repeated five times and the results were averaged. Coefficients of variation (CV) of the results were less than 7%.bAll immobilized samples were made using 1.2 g/L LP solution, and the CLEA-LP was prepared using 0.1% GA crosslinking.

CLEAs-CT and adsorbed-CT, respectively. This resultsuggests that the CT autolysis during immobilizationcontributes to the reduced activities of immobilized CT.Moreover, the number of active sites of CLEAs-CT washigher than that of adsorbed-CT, implying that the CTcrosslinking helps to reduce the irreversible inactivation viaCT autolysis. It is also anticipated that the CT autolysis canresult in a serious mass transfer limitation since theautolyzed products of CT molecules would be denaturedand occupy a lot more space in the pores of HMMS.

To exclude these adverse effects of autolysis on thedetermination of CLEA activity, we prepared the CLEA of anon-proteolytic enzyme, LP, and investigated the activity(Table III) and stability (Fig. 5) of the CLEA-LP. The CLEAapproach improved both loading and stability of LP in asimilar way to CLEA-CT. More specifically, 0.1% GAtreatment improved the LP loading from 13.0% (w/w) ofadsorbed LP to 16.3% (w/w) of CLEA-LP and negligibledecrease of LP activity was observed under rigorous shakingfor 2 weeks while both free and adsorbed LP showed arigorous activity decrease under the same condition. Thespecific activity of CLEA-LP was calculated to be 0.23 mmol/min per mg of LP, which corresponds to the 30% of that of

Figure 5. Stability of CLEA-LP_HMMS, adsorbed-LP_HMMS, and Free-LP in a

shaking condition (200 rpm) at 228C. Initial LP solution of 1.2 g/L and 0.1%

glutaraldehyde was used for the vivid stability enhancement. The relative activity

(%) represents the ratio of residual activity to initial activity of each sample.

[Color figure can be seen in the online version of this article, available at http://

www.interscience.wiley.com.]

free LP (0.78 mmol/min per mg of LP). The decrease of 70%activity in a form of CLEA-LP can be explained by thestructural perturbation of LP structure due to GA cross-linking and the mass transfer limitation of substrate.However, the activity ratio of CLEA-LP to free LP (30%) ismuch higher than that of CLEA-CT to free CT (5%).Considering the fact that the CT can be autolyzed while theLP cannot, the rigorously lowered activity of CLEA-CT (5%)can be ascribed to the CT autolysis and its adverse effect onthe mass transfer limitation of substrate in HMMS.

Conclusions

We developed enzyme reactors in nanometer-scale pores ofHMMS. By crosslinking enzymes within mesocellular poresof a uniquely designed mesoporous material (HMMS), theresulting enzyme aggregates were successfully retained in thepores, providing high loading capacity. Using this ship-in-a-bottle approach, greatly improved stability was achievedwith model enzymes, CT and LP, mainly due to an efficientprevention of enzyme leaching. Since this ship-in-a-bottleapproach of CLEAs in HMMS is very simple and effective forenzyme stabilization, it offers great potential to expand toany other enzymes and any other nano-structured matricesfor the development of stable enzyme system in manyenzymatic applications.

This work was supported by grant No. (R01-2004-000-10293-0) from

the Basic Research Program of the Korea Science & Engineering

Foundation, and partly by the Brain Korea 21 project of the Ministry

of Education. Jungbae Kim thanks U.S. Department of Energy (DOE)

LDRD funds administrated by the Pacific Northwest National Labora-

tory, DARPA/MTO (Contract DE-AC05-76RL01830), and the DOE

Office of Biological and Environmental Research under the Environ-

mental Management Science Program. The research was performed in

part at the W.R. Wiley Environmental Molecular Sciences Laboratory,

a national scientific user facility sponsored by the U.S. Department of

Energy’s Office of Biological and Environmental Research and located

at Pacific Northwest National Laboratory.

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DOI 10.1002/bit

crosslinked enzyme aggregates in hierarchically-ordered mesoporous silica: a simple and effective...

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