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Journal of Biotechnology 125 (2006) 395–407

Chemically surface modified gel (CSMG): An excellentenzyme-immobilization matrix for industrial processes

Allan E. David a,b, Nam Sun Wang a, Victor C. Yang c, Arthur J. Yang b,∗

a Department of Chemical Engineering, A. James Clark School of Engineering,University of Maryland, College Park, MD 20742, United States

b Industrial Science and Technology Network (ISTN) Inc., York, PA 17404, United Statesc Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan,

Ann Arbor, MI 48109-1065, United States

Received 24 October 2005; received in revised form 10 February 2006; accepted 13 March 2006

bstract

Invertase from S. cerevisiae has been immobilized on porous silica matrix, formed using sol–gel chemistry, with surface area ofpproximately 650 m2/g. The co-condensation of silica sol with 3-aminopropyl(triethoxy)silane produced an amino-chemicallyurface modified silica gel (N-CSMG) with a very high ligand loading of 3.6 mmol/g SiO2; significantly higher than commerciallyvailable matrices. Surface amine groups were activated with glutaraldehyde to produce GA-N-CSMG, and invertase covalentlyttached by the aldehyde.

Invertase was used as a model enzyme to measure the immobilizing character of the GA-N-CSMG material. Using anptimized immobilization protocol, a very high loading of 723 mg invertase per gram GA-N-CSMG is obtained; 3–200-foldigher than values published in literature. The reproducible, immobilized activity of 246,000 U/g GA-N-CSMG is also greaterhan any other in literature. Immobilized invertase showed almost 99% retention of free enzyme activity and no loss in catalyticfficiency. The apparent kinetic parameters KM and VM were determined using the Michealis–Menten kinetic model. KM of

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he free invertase was 1.5 times greater than that of the immobilized invertase—indicating a higher substrate affinity of themmobilized invertase. These findings show considerable promise for this material as an immobilization matrix in industrialrocesses.

2006 Elsevier B.V. All rights reserved.

eywords: Enzyme immobilization; Invertase; Amino-functionalized silica

∗ Corresponding author at: Industrial Science and Technology Net-ork (ISTN) Inc., CYBER Center, 2101 Pennsylvania Avenue, York,A 17404, United States. Tel.: +1 717 843 0300;ax: +1 717 843 0705.

E-mail address: [email protected] (A.J. Yang).

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168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jbiotec.2006.03.019

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. Introduction

The two major driving concerns in an industrialcale process are how to lower the unit cost and toncrease the unit production per fixed time. For prod-

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dx.doi.org/10.1016/j.jbiotec.2006.03.019

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96 A.E. David et al. / Journal of

cts obtained from an enzymatic reaction, the firsttep towards achieving these goals is enzyme immo-ilization. Immobilization would allow the use of thenzyme in multiple batches thereby reducing the costsf unit production. In addition, enzyme-immobilizedesins or particulates could easily be packed into aed or a column for flow through reactions. Such aeactor would achieve a high enzyme concentrationuring the reaction process and also avoid the time-onsuming step of enzyme separation from the reactionroducts.

A variety of matrixes have been used as sup-ort materials for enzyme immobilization (Girelli andattei, 2005). Many of these materials, however, fall

nder the category of so-called “soft gels” due toheir low mechanical strength, rendering them unfitor most of rather vigorous industrial processing pro-edures (Sakaguchi et al., 2005). The use of silica-ased gels for enzyme immobilization has receivedncreased attention for industrial manufacturing ofnzyme-processed products (Pierre, 2004; Ho et al.,004; Blanco et al., 2004; Fonseca et al., 1993). Sil-ca gels offer a number of advantages over “softels” for use in industrial processes. First, the signif-cantly higher mechanical strength would allow for

much wider range of operating pressures; as evi-enced by their preferential use in the preparationf high performance liquid chromatography (HPLC)Xu et al., 2004; Majors, 2003; Cabrera, 2004). Addi-ionally, silica gels possess relatively higher thermalnd chemical stabilities under conditions involvedn industrial processing, and their inert nature alsoenders them resistant to microbial contaminationr degradation (Pang et al., 2002; Nefedov, 1992).ost critically, silica gels provide exceedingly high

urface areas and porosity, which in turn can besed to enhance enzyme loadings and accessibil-ty.

Invertase is a widely used enzyme in the foodndustry for the production of sweeteners used ineverages, jams, and as artificial honey (Cheetham,995). It catalyzes the cleavage of the �-1,4-glycosidiconds of sucrose, producing glucose and fructoseKhobragade and Chandel, 2002) via the following

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eaction: sucrose + H2O → glucose + fructose. Inver-ase has a molecular weight of 270 kDa (Lampen,971; Kupcu et al., 1991) and an isoelectric pointetween 3.4 and 4.4 (Righetti and Caravaggio, 1976).

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nology 125 (2006) 395–407

o co-factors are required for activation of invertasehile its activity is inhibited by the presence of iodine,g+, Zn2+, and Hg2+ (Lampen, 1971; Goldstein andampen, 1975; Huttl et al., 1999). These properties, asell as the commercial significance, make invertase an

deal model to study enzyme immobilization onto theilica-based support material. In addition, invertase haseen immobilized onto a number of supports includ-ng clay (Sanjay and Sugunan, 2005), methacrylate-ased polymers (Cirpan et al., 2003; Bayramoglu etl., 2003), Sepabeads (Torres et al., 2002), celite andolyacrylamide (Mansour and Dawoud, 2003), men-hyl ester (Kiralp et al., 2003), organosilanes (Airoldind Monteiro, 2003), and agarose (Fuentes et al.,004). Hence, the selection of invertase would alsollow for comparison of the benefits of utilizing silicaels as the support versus a variety of other possibleaterials.While enzyme immobilization has been studied for a

umber of years, the appearance of published researchnd review papers (Hartmann, 2005; Kallenberg et al.,005; Pierre, 2004; Krajewska, 2004; van de Velde etl., 2002; Boller et al., 2002) indicate a continued inter-st in this area; more than 550 enzyme immobilization-elated papers have been published in 2005. This isikely due to the known benefits of enzyme immo-ilization and the desire to improve immobilizationatrices and methods. Current immobilization tech-

iques provide very low enzyme loadings, relative tovailable surface areas, and low immobilized enzymectivity, relative to the enzyme’s native activity. In thisaper, we describe a method and material for sig-ificantly improving the enzyme loading while alsoetaining enzyme activity. Silica gels containing func-ional amino groups on the surface, nicknamed N-SMG (i.e. amino-chemically surface modified gel),ere prepared by using a sol–gel manufacturing pro-

ess. Invertase was then immobilized onto N-CSMGsing an optimized, well-established glutaraldehydeoupling method. The immobilization process, enzymeoadings, and enzymatic/kinetic properties of the N-SMG-immobilized invertase were thoroughly char-cterized by utilizing a variety of physical and chemicalethods. To validate the benefits of utilizing N-CSMG

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s the support for immobilized enzymes, findings dis-overed in this paper were compared with literatureeports on invertase immobilized onto various matri-es.

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. Materials and methods

.1. Materials

Invertase (Grade VII—measured activity of 181 U/g protein at 25 ◦C) from baker’s yeast, glutaraldehyde

25 wt.% in water), (3-aminopropyl)triethoxysilane98% pure), and sucrose were purchased from Sigma-ldrich (St. Louis, MO) and used as supplied. Sodium

ilicate (N-type) was purchased from PQ CorporationValley Forge, PA). All other reagents were of analyti-al grade, and water was distilled and deionized.

.2. Methods

.2.1. Synthesis of amino-chemically surfaceodified gel (N-CSMG)N-CSMG was synthesized by using sol–gel man-

facturing procedures. In brief, silicic acid was pro-uced from sodium silicate using an ion-exchangerocess developed at ISTN Inc. (York, PA). The ion-xchange, accomplished with anionic Amberlite resin,xchanges sodium ions for hydrogen to produce a lowonic strength, silica sol with reactive silanol groups–Si–OH). Formation of N-CSMG was completed byddition of ethanol and 3-aminopropyltriethoxy-silaneAPTES) to the silicic acid. Addition of the basicPTES to silicic acid increases the solution pH and

nduces the gelation. Gelation occurs through the con-ensation of silanol groups. Co-condensation betweenilica sol and APTES produces silica gels with surface-odified functional amino groups. The monolithic

el was mechanically broken into particles of 11 �mverage diameter (ranging from 7 to 38 �m; as mea-ured by using a CEDEX particle size analyzer with40 �m measurement limit). The gel particles were

hen washed with ethanol, vacuum filtered, and washedxtensively with deionized water. The final filtered gelarticles were stored at 7 ◦C until use for a maximumeriod of 2 weeks.

.2.2. Glutaraldehyde (GA) activation of-CSMG to produce glutaraldehyde-activated

ilica gel (GA-N-CSMG)


To activate N-CSMG with glutaraldehyde, the gelas suspended in glutaraldehyde solution at a ratiof 20 mL solution per gram N-CSMG. The suspen-ion was magnetically stirred at room temperature for

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nology 125 (2006) 395–407 397

4 h, vacuum filtered to remove glutaraldehyde fromhe solution, and then washed extensively with deion-zed water. The final filtered gel was stored at 7 ◦C untilse.

.2.3. Immobilization of invertase onA-N-CSMGTo immobilize invertase, 500 mg of GA-N-CSMG

ere weighed into a 50 mL centrifuge vial. Twentyilliliters of invertase (in 50 mM acetate buffer at pH

.5) were introduced to the vial, and the suspensionas magnetically stirred at 7 ◦C for at least 48 h. The

uspension was centrifuged at 10,000 × g for 10 min,nd the supernatant was then removed.

The invertase-immobilized gel was washed threeimes with 20 mL of 100 mM acetate buffer (pH 4.5)nd centrifuged between each wash. Three washes wereound to be adequate for removal of physically adsorb-d invertase on the gels, as demonstrated by a significa-t decline of the enzyme activity in the supernatant. Thenal wash was carried out for 4 days under vigoroustirring, and <1% of the initial invertase activity wasound to remain in the supernatant. This finding furtheruggested that the immobilized invertase was quite sta-le, and no significant leaching of the enzyme occurred.he thoroughly washed gels were suspended in 20 mLf 100 mM acetate (pH 4.5) and stored at 7 ◦C until use.

.2.4. Determination of enzyme activityInvertase activity was determined by monitoring the

ydrolysis of 30 mL sucrose (50 mg/mL) in 50 mMuffer in a magnetically stirred, thermostated vessel.he activity was measured by using the dinitrosali-ylic acid (DNS) assay method (Miller, 1959). Briefly,t various times, 500 �L aliquots of the reaction solu-ion were withdrawn and added to 5 mL of solutionontaining 10 mg/mL DNS to quench the reaction.he sample vials were capped and heated in boilingater for 30 min prior to addition of 1 mL sodium tar-

rate (40 wt.%) to stabilize the color. Time-dependenthange of glucose concentrations was then determinedy measuring the absorbance at 575 nm, and invertasectivity was estimated from the initial rate measure-ents of the reaction kinetics. Enzyme activity was

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xpressed in units per gram (U/g), where one unit wasefined as the production of 1 �mol glucose product perinute (�mol/min). All activity tests were conducted

n triplicate.

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.2.5. Optimization of glutaraldehydeoncentration

The optimum glutaraldehyde concentration wasetermined by adding 20 mL of glutaraldehyde solu-ions to vials containing 1 g of wet weight of N-CSMG.fter allowing the reaction to take place for 24 h at

oom temperature, the gel was thoroughly washed witheionized water and vacuum filtered. To 500 mg (weteight) of activated N-CSMG, 20 mL of 2 mg/mL

nvertase in 50 mM acetate (pH 4.5) were added, andmmobilization was allowed to take place at 7 ◦C for8 h. The gel was then washed several times, suspendedn 100 mM acetate buffer (pH 4.5), and measured fornvertase activity at room temperature. Glutaraldehydeoncentration was varied from 0 to 10 wt.%.

.2.6. Optimization of invertase concentrationThe optimum concentration for immobilization of

nvertase was determined by adding 20 mL inver-ase solution at various concentrations ranging from–20 mg/mL to 500 mg of the above optimized GA-N-SMG.

.2.7. Determination of immobilized invertasetability

The stability of immobilized invertase, stored at◦C, was determined by periodically removing sam-les, over a 1 month period, and testing for enzymaticctivity according to the procedure detailed above.

The operational stability was determined by pack-ng 60 mg of immobilized invertase, taken after the

month storage period above, into a glass columnf 1.0 cm diameter. The packed enzyme was washedith 60 mL of 0.1 M acetate, pH 4.5, at a flow rate of.0 mL/min. This was then followed by introduction of50 g/L sucrose solution, in 50 mM acetate (pH 4.5),

t a flow rate of 1.0 mL/min. The exit stream was ana-yzed for glucose concentration by the DNS methodver a period of 6 h.

. Results and discussions

.1. Characterization of N-CSMG material


One advantage that nanoporous silica materialsrovide, compared to other supporting matrices fornzyme immobilization, is the significantly larger sur-

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nology 125 (2006) 395–407

ace area. The surface area of the optimized GA-N-SMG was determined, on a Quantachrome Nova

Model 1200), by analysis of nitrogen adsorptionsotherms using the BET method (Brunauer et al.,938). It should be noted that silica-based materialsould experience a significant degree of shrinkage uponrying, which would in turn reduce the surface area.o this regard, the supercritical drying method, ini-

ially developed by Kistler (1931, 1932, 1937) and laterodified by Tewari et al. (1985), was followed to avoid

hrinkage caused by recession of the liquid/vapor inter-ace into the gel. Supercritical CO2 was used to dryhe optimized GA-N-CSMG material prior to surfacerea analysis. This treatment allowed for obtaining arue surface area measurement of the material in itset state. Following out-gassing under vacuum for 2 h

t 100 ◦C, the surface area of the optimized GA-N-SMG was determined to be approximately 650 m2/g.ecause all surface modification reactions, enzyme

mmobilization, and enzymatic activity measurementsere conducted using the wet gel, this surface area is

ruly characteristic of the material tested.

.2. Optimization of glutaraldehyde concentration

Glutaraldehyde is a bifunctional crosslinker com-only used to couple components with amino func-

ional groups. Activation of amino-modified silica with0 mL glutaraldehyde solutions of 0, 0.31, 0.63, 1.25,.5, 5, and 10% (w/w) concentrations yielded relativemmobilized invertase activities of 32(±0), 100(±0),00(±0), 94(±5), 89(±6), 100(±0), and 98(±4) per-ent, respectively. As seen by this data, no significantncrease in immobilized enzyme activity was seen withncreasing glutaraldehyde concentrations up to 10%.he lowest, yet effective, glutaraldehyde concentration

n achieving maximum invertase immobilization wasound to be 0.31% (w/w). The lack of significant varia-ion of immobilized enzyme activity seen with even theowest concentration is likely due to the abundance oflutaraldehyde in solution compared to available aminoroups on the silica.

A 20 mL volume of the 0.31% (w/w) solutionontained approximately 0.625 mmol glutaraldehyde


hile a gram of wet N-CSMG, with a dry weight of0 mg, contained about 0.35 mmol of amino groups.ven with this low glutaraldehyde concentration, thereas approximately a 1.8 molar ratio of glutaraldehyde

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o surface amino groups. Increasing the glutaraldehydeoncentration further had no effect on the immobilizednzyme activity because the system was already satu-ated.

It is interesting to note that N-CSMG withouthe glutaraldehyde treatment yielded a relatively highmmobilized enzyme activity of approximately 32%.his immobilization may occur through the formationf salt-bridges between the basic N-CSMG surface andhe side-chains of the acidic amino acids in invertase;

well established interaction seen in acidic proteinsKing et al., 1991; Dunten et al., 1993; Sapse et al.,002) such as invertase (pI = 3.4–4.4).

.3. Optimization of invertase concentration

The optimum invertase concentration for immo-ilization was determined by varying its concentra-ion from 0 to 20 mg/mL. For each concentration,0 mL of invertase solution (in 50 mM acetate, pH 4.5)as added to 500 mg (wet weight) of glutaraldehyde-

ctivated N-CSMG.As shown in Fig. 1, the highest immobilized enzyme

ctivity was achieved at 10 mg/mL invertase concen-


ration, with no further increase in immobilized activ-ty being observed at higher enzyme concentrations.owever the immobilization yield, calculated from the

atio of immobilized activity versus the activity in the

ig. 1. Effects of invertase concentration on its immobilization.nvertase solutions of varying concentrations were added to GA--CSMG according to the procedures described in Section 2. �

epresents the immobilized activity whereas � represents the immo-ilization efficiency. Experiments were conducted in triplicate at0 ◦C. The error bars indicate the standard deviation.

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nology 125 (2006) 395–407 399

riginal solution, was only 23% when using 20 mLf the 10 mg/mL invertase solution with 500 mg ofet glutaraldehyde-activated N-CSMG. For this rea-

on, the immobilization process was further optimizedy varying the volume of the 10 mg/mL invertase solu-ion from 1 to 20 mL. Results, not shown, indicatedhat a solution volume of 10 mL yielded the maximummmobilization efficiency (71%) as well as 90% of the

aximum immobilized activity, observed when 20 mLf 10 mg/mL invertase solution was used. All subse-uent invertase immobilization was therefore carriedut by using 500 mg wet GA-N-CSMG and 10 mL ofhe 10 mg/mL invertase solution.

.4. Effect of pH on invertase activity

Changing pH is known to affect the stability of annzyme, as most enzymes display a strong dependencef activity on pH. The pH-dependent activity profilesf both free and immobilized invertase are shown inig. 2. Free invertase showed a maximum activity atH 5.0 whereas the immobilized invertase was mostctive at pH 4.0. This shift of optimum pH towards ancidic value for immobilized invertase is because the


nzyme experiences a greater local pH than that of theulk solution due to the basic nature of the functional-zed silica surface; with ionic groups such as the aminoroups on the gel surface there is an uneven distribution

ig. 2. Effects of pH on invertase immobilization. The solutionH of either invertase or GA-N-CSMG-immobilized invertase weredjusted according to the procedures described in Section 2. �epresents the free invertase whereas � represents the GA-N-CSMG-mmobilized invertase. Experiments were conducted in triplicate.he error bars indicate the standard deviation.

4 Biotechnology 125 (2006) 395–407

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Table 1Comparison of activation energy and pre-exponential factor of freeand immobilized invertase

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Pre-exponentialfactor (U/g protein)

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f hydrogen ions between the surface and bulk solutionChaplin and Bucke, 1992; Shuler and Kargi, 1992). Aigher local pH for the immobilized invertase couldlso explain the greater activity loss seen in Fig. 2 formmobilized invertase relative to free invertase, whenhe pH was raised above 4.5. Also immobilization mayave caused a conformational change of the enzyme,hich in turn results in a higher invertase activity at a

ower pH. Invertase is a multimeric enzyme with a min-mum active state as a dimer but it also exists as larger

ultimers under certain pH and concentration condi-ions (Torres et al., 2002). It is possible that the requiredimer state is unfavorable under the conditions whereoss of immobilized enzymatic activity, relative to freenvertase, is observed.

.5. Effect of temperature on invertase activity

The effect of temperature on both free and immobi-ized invertase activity was determined by measuringhe hydrolysis of sucrose at temperatures ranging from5 to 75 ◦C. As seen in Fig. 3, both the immobilizednd free enzymes exhibited a very similar temperaturerofile, implicating that immobilization of invertase onA-N-CSMG did not significantly alter the enzyme’s

emperature stability.Arrhenius plots, based on data observed in Fig. 3,

howed a linear relationship, which indicates an


ncrease in enzyme activity with increasing tempera-ure, for both the immobilized and free invertase upo 55 ◦C. As the temperature was further increased,owever, enzyme denaturization occurred and a cor-

ig. 3. Effect of temperature on initial activities of free (�) andmmobilized (�) invertase.

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esponding drop in enzyme activity was demonstrated.he slopes of the lines, within the region where tem-erature dependent denaturization did not occur, weresed to determine the activation energy of the enzymeediated reaction using Eq. (1):

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RT(1)

Results in Table 1 show that the immobilizednvertase displayed a reduced activation energy of5.4 kJ/mol comparing to the value of 31.3 kJ/mol forree invertase. Reports from literature demonstratedhat a decrease in activation energy can be correlatedo intraparticle diffusion of the substrate (Miyamoto etl., 1973). The appearance of the diffusion effects onhe activation energy provided an indication that themmobilization was occurring within a porous matrix.n the case of a nonporous matrix, the activation energyf both the free and immobilized enzyme would be inloser agreement with each other (Bahar and Tuncel,002). It is known that the pre-exponential factor, alsoalled the frequency factor, is proportional to the prob-bility of a collision resulting in a substrate to producteaction. As seen in Table 1, this factor is very similaror both the immobilized and free invertase.

.6. Michaelis–Menten kinetic parameters

The Michaelis–Menten kinetic parameters, appar-nt values in the case of immobilized enzyme, of bothree and immobilized invertase were determined byeasuring the initial reaction rates at various initial

ubstrate concentrations. Typical Lineweaver–Burklots for both the free and immobilized invertase wereenerated; the kinetic parameters obtained from theselots are summarized in Table 2.

The quantity K−1m is a measure of the stability of the

S complex or, in other words, affinity of the enzymeor its substrate. As seen in Table 2, the immobilizednvertase exhibited a higher affinity for sucrose (lower

A.E. David et al. / Journal of Biotechnology 125 (2006) 395–407 401

Table 2Michaelis–Menten kinetic parameter of free and immobilized invertase

Vmax (U/mg) Km (mol/L) Catalytic efficiency (Vmax/Km)

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SKaCA(1ihsnvpiin other spectra, indicating a successful activation ofthe amino groups on N-CSMG by glutaraldehyde andconsumption of the groups during invertase immobi-lization. A peak at 1647 cm−1, seen in the spectra of

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ree (8.2 ± 1.0) × 102

mmobilized (6.0 ± 0.6) × 102

m gives a larger K−1m ) than free invertase while the

aximum reaction rate, Vmax, was decreased comparedo that of the free enzyme. A catalytic efficiency, whichs reflected by the ratio of Vmax over Km, was foundo be similar for both the immobilized and free inver-ase. The slight increase in substrate affinity was ableo compensate for a decreased Vmax for immobilizednvertase, providing a catalytic efficiency equal to thatf the free enzyme.

The efficiency factor, η, was calculated from theaximum reaction rates of the immobilized invertase

ver that of the free invertase:

= νimmob

νfree= 0.73 (2)

here νimmob was the reaction rate of the immobilizednvertase and νfree that of free invertase. From this cal-ulation, GA-N-CSMG provided an efficiency factor of.73 for the immobilization of invertase. It was reportedhat as the effectiveness factor decreases below unity,he measured activation energy approaches the arith-

etic mean of the activation energies of diffusion andeaction (De Whalley, 1964).

There are several reasons that can explain the differ-nce in behavior of the free and immobilized invertase.irst, the immobilized invertase resides in an environ-ent that is quite different from that of the free enzyme

n the bulk solution. The dependence of immobilizednvertase on diffusion of substrate into the matrix inte-ior is also quite different from free invertase, whichnteracts freely with the bulk solution. In addition, thettachment of invertase to GA-N-CSMG likely causesome change in conformation or increase in steric hin-rance. A conformational change may explain why them of immobilized invertase is reduced to 67% of the

ree enzyme, indicating a higher affinity of the immobi-ized invertase for sucrose compared to free invertase;
he change in conformation may have opened or moreavorable positioned the active site. Although in mostases immobilized enzyme would show a decrease inubstrate affinity due to conformation changes (Bahar
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nd Tuncel, 2002), our results showed that immobiliza-ion of invertase on GA-N-CSMG actually increased itsubstrate affinity.

.7. FT-IR analysis of invertase immobilization

Infrared spectra were obtained using a Perkin-Elmerpectrum BX to analyze pellets formed with mixedBr and solid samples (1 wt.%). Shown in Fig. 4

re spectra obtained for unmodified silica gel, N-SMG, GA-N-CSMG, and the immobilized invertase.s expected, the C–H stretching vibration frequency

Airoldi and Monteiro, 2000; Monteiro and Airoldi,999) at 2936 cm−1 was seen in all spectra, display-ng contributions from the organosilane, glutaralde-yde, and enzyme, except in the spectra for unmodifiedilica, which does not contain any organic compo-ent. Furthermore, the corresponding simple bendingibrations are seen between 1500 and 1300 cm−1. Theresence of free aldehyde groups (at 1718 cm−1) wasdentified in the spectrum of GA-N-CSMG but not

ig. 4. FT-IR analyses of: (a) unmodified silica gel; (b) N-CSMG;c) GA-N-CSMG; (d) immobilized invertase and (e) free invertase.

4 Biotechnology 125 (2006) 395–407

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Loading

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A-N-CSMG and immobilized invertase, presumablyepresents the imine (N C) Schiff-base produced byhe amine-glutaraldehyde reaction; this peak appearsn each of the spectra due to the presence of primaryH bending or contributions from moisture. The peak

een at 1550 cm−1 in all spectra except for pure silicaould be attributed to primary amine salt –NH3

+ (Lin-ien, 1991). The spectra below 1300 cm−1 is not shownecause of the strong bands appearing in the rangef 1250–1000 cm−1 related to the Si–O–Si asymmet-ic stretching vibration and the Si–OH bending bandsppearing between 900 and 750 cm−1 (Coates, 2000;osa et al., 2000).

.8. Thermogravimetric analysis (TGA) ofmmobilized invertase loading

TGA analysis, performed on a Perkin-Elmer TGA-with samples heated at a rate of 1.5 ◦C/min in an

ir atmosphere, was used to confirm the high enzymeoadings on CSMG as observed from the high immobi-ized invertase activity. Fig. 5 shows the TGA profilesbtained for silica (silicic acid gelled without sur-ace modification), N-CSMG (amino-modified silica),A-N-CSMG (N-CSMG activated with glutaralde-yde), and the immobilized invertase (invertase immo-


ilized onto GA-N-CSMG). As the reactor tempera-ure was increased, organic groups within the silicael were vaporized and carried away on a stream ofir. The final residual weight was therefore an excel-

ig. 5. Thermogravimetric profiles for silica gel without modifi-ation (silica); amino-modified silica (N-CSMG); glutaraldehyde-ctivated silica (GA-N-CSMG) and invertase-immobilized silicaimmobilized invertase).

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lutaraldehyde 0.634 7.5nvertase 1.39 5 × 10−3

ent index to monitor the abundance of silicon dioxideSiO2).

Loadings (g/g SiO2) of the various componentsere then determined from Fig. 5 by comparison of

he residual weights; the molar loadings were esti-ated by using the known molecular weights of

hese components. Table 3 provides a summary ofhese results. As assessed by thermogravimetric anal-sis, the N-CSMG material contained a loading ofpproximately 3.6 mmol amino functional groups perram of silica. This loading was significantly higherhan the 1.0 mmol/g loading observed from existingommercially available products (e.g. Sigma-Aldrich364258), and allowed for much higher loadings forroteins. The achieved amino loading is almost 88%f the 4.16 mmol/g SiO2 maximum theoretical load-ng calculated based on the material’s surface areand an APTES footprint of 50 A2 (Vansant, 1995).t was expected that glutaraldehyde would react, atost, in a one-to-one molar ratio with surface amino

roups. However, the TGA data shows that glutaralde-yde/amino ratio is actually 7.5:3.6 mmol/g SiO2—aatio of more than 2:1. The dimerization of glutaralde-yde has been shown to occur in aqueous alkalineolutions (Tashima et al., 1991) and, in this case, mayave been precipitated by the basic nature of the gel.s seen, N-CSMG was able to achieve a very high

nvertase loading, with a maximum loading of 1.39 gnvertase per gram of SiO2 observed. This invertaseoading translates into a loading of 723 mg invertaseer gram of the GA-N-CSMG matrix.

The invertase loading obtained with GA-N-CSMGas significantly higher than other published results.
comparison of the GA-N-CSMG loading with var-
ous other materials is presented in Table 4. With aoading of 723 mg/g matrix, GA-N-CSMG was ableo immobilize a three-fold greater weight of invertase

A.E. David et al. / Journal of Biotechnology 125 (2006) 395–407 403

Table 4Comparison of invertase immobilization loading and the retained activity of GA-N-CSMG with other materials

Carrier Invertase loading (mg/g matrix) Retained activity (%) Reference

GA-N-CSMG 723 98.7 ± 4.1Organosilane 3.51 – Airoldi and Monteiro (2003)Sepabeads – 90 Mateo et al. (2003)Pectin – 57 Gomez and Villalonga (2000)Celite 3 92 Mansour and Dawoud (2003)PPL

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lpTislmatrix, the highest activity in literature was 11,500 U/g(Vicente, 2000b); again a more than 20-fold reductionof the loading obtained by using the N-CSMG matri-ces.

Table 5Loading efficiency based on surface coverage of GA-N-CSMG byimmobilized invertase

Molecular weight 270000 DaStokes radius 5.63 nm


olyacrylamide 10oly(p-chloromethylstyrene) beads 19actam-amide graft copolymer 225

han the material with the next highest loading, i.e.he lactam-amide graft polymer with a loading of25 mg/g. However, while the lactam-amide graft poly-er immobilization exhibited only 15% retention of

he enzyme activity, almost 99% of free invertasectivity was retained by invertase immobilized onA-N-CSMG; specific activities were 345.4 ± 11.6

nd 340.9 ± 8.8 U/mg protein for free and immobilizednvertase, respectively, at pH 4.5 and 45 ◦C.

The efficiency of invertase immobilization on GA--CSMG was also analyzed by measuring the degree of

urface coverage. As noted, the Stokes radii of proteinss generally determined from sedimentation experi-

ents using the Stokes–Einstein equation (Gosting,956):

= kT

6πηD(3)

here a is the Stokes radius in A, k the Boltzmannonstant, T the absolute temperature, η the viscosity,nd D is the diffusion coefficient. Uversky has derivedcalibration curve for the dependence of a protein’stokes radius on the molecular weight based on pub-

ished results (Uversky, 1993):

og(RS) = −(0.254 ± 0.002)

+(0.369 ± 0.001) log(MW) (4)

here RS is the Stokes radius in A and MW is therotein molecular weight in Daltons. Applying thisquation to the 270 kDa invertase and 649 m2/g surfacerea for GA-N-CSMG, the loading efficiency based
n surface coverage was estimated to be 24.7% (seeable 5).
Although a relatively high loading of invertaseas achieved on CSMG compared to other published

MGTAL

8180 Bahar and Tuncel (2002)15 De Queiroz et al. (2002)

esults, there still appears to be considerable room forurther improvement. Based on the molecular footprint,alculated from the Stokes radius, and the measuredurface area, a theoretical maximum loading of 2.93 gnvertase per gram GA-N-CSMG would be achievablesee Table 5). To this end, the obtained loading of.723 g/g GA-N-CSMG was merely less than 25% ofhe theoretically predicted maximum loading. Reasonshat may account for this low loading efficiency includehe inaccessibility to surface areas by the enzyme due tohe presence of small micropores as well as the block-ge of pore entrances by the gel networks (Baruque etl., 2001; Raman et al., 1993). In addition, multi-pointttachments between the enzyme and surface wouldlso reduce the loading.

Table 6 provides a comparison of the immobi-ized invertase activity of GA-N-CSMG with someublished results under similar reaction conditions.he highest activity seen in literature for immobilized

nvertase was 36,000 U/g for immobilization on corntover (Monsan et al., 1984); a result almost seven-foldower than that of the GA-N-CSMG. For a silica-based

olecular footprint 99.4 nm2

A-N-CSMG surface area 649 m2/gheoretical loading per g GA-N-CSMG 2.93 g/gctual loading per g GA-N-CSMG 0.723 g/goading efficiency 24.7%

404 A.E. David et al. / Journal of Biotechnology 125 (2006) 395–407

Table 6Comparison of GA-N-CSMG-immobilized invertase activity with various other matrix materials

Matrix Particle size (�m) Activity (U/g matrix) Temperature (◦C),pH, [S] (M)

Reference

GA-N-CSMG 7–200 246000 45, 4.5, 0.146Corn grits 100–200 36000 40, 4.5, 0.400 Monsan et al. (1984)Porous silica – 11500 45, 4.5, 0.146 Vicente (2000a)Aminopropyl silica – 3700 45, 4.5, 0.146 Rosa et al. (2000)PM

3i

bFpSotsS

ttdliraf

Fp


olystyrene 300 3050agnetic silica 20–45 469

.9. SEM analysis of GA-N-CSMG after invertasemmobilization

SEM was performed to further monitor the immo-ilization process. As seen from the SEM images inig. 6, there was an evolution of the particle mor-hology of CSMG with each of the processing steps.urface modifications, in combination with the effects
f the mechanical stirring used, created silica par-icles with a rougher appearance and finer particleize. A significant change in appearance was seen inEM images from N-CSMG to invertase immobiliza-
sepi

ig. 6. SEM images of (a) N-CSMG, (b) GA-N-CSMG, (c) GA-N-CSMG-ores.

25, pH 4.5, 0.146 Mansfeld and Schellenberger (1987)55, 4.5, 0.292 Goetz et al. (1991)

ion. While N-CSMG exhibited a smooth surface onhe micron scale, the invertase-immobilized CSMGisplayed a significant degree of porosity. This wasikely created through the breaking of GA-N-CSMGnto finer particles by mechanical stirring and thene-interacting to form cluster structures. These inter-ctions could occur between aldehyde groups of dif-erent particles or through the immobilization of a
ignal enzyme on two different silica particles. Thend result was a material that was not only meso-orous, but also contained larger pores that wouldncrease the diffusion rate of both substrate and
immobilized invertase, and (d) high magnification of GA-N-CSMG

A.E. David et al. / Journal of Biotech

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中国科技论文在线 n

ig. 7. Storage stability: retention of enzymatic activity when storedt 7 ◦C. The errors bars indicate the standard deviation.

roduct towards and from the immobilized enzyme,espectively.

.10. Determination of immobilized invertasetability

Preliminary tests were conducted to determine thetability of the immobilized enzyme. Fig. 7 shows thathe immobilized enzyme can be stored, at 7 ◦C, over

1 month period with no significant loss of enzy-atic activity. In addition, the immobilized invertaseas successfully employed in a packed-bed reactor to

chieve the continuous hydrolysis of sucrose. As shownn Fig. 8, after an initial start-up period of 30 min, theystem reached a continuous level of glucose produc-ion of approximately 0.118 M glucose—an 80% yield
rom the 0.146 M sucrose feed. This constant level ofroduction was maintained over 270 min. Further test-ng is ongoing to determine the long term stability ofnvertase immobilized on N-CSMG.
ig. 8. Operational stability: retention of enzymatic activity whenperated as a packed-bed reactor at room temperature.

afiiisoebfe

A

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nology 125 (2006) 395–407 405

. Conclusion

As a supporting matrix for ligand immobilizationn the industrial manufacturing of chemicals or com-ounds, silica gel offers a number of unmatched advan-ages over other existing support materials. First, theurface of silica gel can be readily modified by chem-cal methods to provide various types of functionalroups for facilitated ligand attachment. Results pre-ented in this paper provide an excellent exampleo justify this statement. Extremely high loading ofnvertase was easily obtained by utilizing the amino-hemically surface modified gel (N-CSMG) product.econdly, the overall surface areas that the silica gelatrices can offer are unparalleled. While the standardepharose CL-6B (6% agarose) particles can providetotal surface area of 25 m2/mL (Suh et al., 2003), theSMG product prepared in the current study can eas-

ly reach the value of 73 m2/mL (calculated based onhe measured surface area of 649 m2/g and density of.113 g/mL); an approximately three-fold increase. Asconsequence, the invertase loading achieved on N-SMG (i.e. 0.723 g/g matrices) was 3–200-fold higher

hat those reported in the literature on other supportaterials. It should be pointed out that the ultimate

nvertase loading on N-CSMG can be dramaticallyncreased, as our theoretical assessment suggested thathe current loading was only 25% of the potential max-mum loading. Thirdly, the silica gel can be easilyabricated to provide desirable morphology (Pang etl., 2002), pore structures, and micro-channels to allowor substrate–ligand interaction. Last but not least, sil-ca gel is mechanically stable and chemically inert, ands therefore environmental- and solvent-friendly forndustrial manufacturing and processing. Results pre-ented in the paper indicated that a complete recoveryf invertase activity could be attained under very benignxperimental conditions. These vastly improved andeneficial factors render N-CSMG an excellent choiceor industrial processing of immobilized ligands ornzymes.

cknowledgements


This work was supported in part by NIH Grants R43K67723 and funds from Industrial Science and Tech-ology Network (ISTN) Inc.

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chemically surface modiﬁed gel (csmg): an excellent enzyme ...€¦ · allan e. davida,b, nam sun...

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