continuous surface modification of silica particles for enzyme immobilization

Advanced Powder Technol., Vol. 14, No. 2, pp. 231–245 (2003)Ó VSP and Society of Powder Technology, Japan 2003.Also available online - www.vsppub.com

Original paper

Continuous surface modi�cation of silica particlesfor enzyme immobilization

SHUHEI SHIOJI 1;¤, MASASHI HANADA1, YASUHIRO HAYASHI 1,KENJIRO TOKAMI1 and HIDEO YAMAMOTO2

1 Department of Material Science, Wakayama National College of Technology, 77 Noshima,Nada-cho, Gobo 644-0023, Japan

2 Department of Bioengineering, Faculty of Engineering, Soka University, 1-236 Tangi-cho,Hachioji 192-0003, Japan

Received 8 July 2002; accepted 30 August 2002

Abstract—A novel reaction process for surface modi�cation of ceramic particles is investigatedfor enzyme immobilization. The surface modi�cation of the support particles can be carried outcontinuously in a �ow reactor. The proposed continuous surface modi�cation process (CSMP) isadvantageous for the handling of particles in the catalyst preparation process. Using this reactionmethod, the surface of silica particles was easily modi�ed for enzyme immobilization. The degree ofsurface modi�cation could be controlled by the reaction conditions and the amount of immobilizedenzyme was also controlled. Lysozyme and catalasewere immobilized on the silica particles modi�edby the CSMP method. The adsorption of lysozyme was studied as an example of enzyme adsorptionon the modi�ed particles. The enzyme activity of prepared particles was tested for decomposition ofH2O2 by immobilized catalase. It was shown that the obtained particles indicated the enzyme activityand CSMP is a useful method for preparing enzyme catalysts.

Keywords: Silica gel; surface modi�cation; surface amination; enzyme immobilization.

NOMENCLATURE

C concentration (mol/dm3)Km Michaelis constant (mol/dm3)kA rate constant of surface amination reaction (m3 /mol/ s)k0 initial rate constant in (5) (1/min)NA number of surface amino groups (nm¡2)r0 initial rate of reaction in (5) (mol/dm3 /min)Tp preheating temperature (±C)Tr reaction temperature (±C)

¤To whom correspondence should be addressed. E-mail: [email protected]

232 S. Shioji et al.

Greek

0 amount of total adsorbed enzyme (g/g)0c amount of chemisorbed (immobilized) enzyme (g/g)0p amount of physisorbed enzyme (g/g)μ reaction time (min)

1. INTRODUCTION

It is well known that immobilization of enzymes is a useful method in order toapply them in industrial bio-reactors. By immobilization on solid particles, enzymescan be easily handled and used as excellent catalysts in the chemical reactions.The preparation of enzyme-immobilized particles is an important process whichdetermines the ef�ciency of the bio-reactor systems. In general, prior surfacemodi�cation of support particles is needed to immobilize enzyme on ceramicparticles. Ordinary surface modi�cation reactions are carried out in solutions, anddrying processes are needed before and after the chemical reactions. Thus, thepreparation of enzyme catalyst particles is a discontinuous process and the handlingef�ciency of the particles becomes low. These factors result in dif�culties in theproduction of homogeneous enzyme catalyst particles on a large scale. In order toincrease the overall ef�ciency of the biochemical process, improvements are neededfor the preparation of enzyme catalysts.

Enzymes are immobilized on silica particles such as silica gel, quartz and glassas well as organic materials. The use of these inorganic particles is advantageousin terms of chemical stability during various reactions. The surfaces of support par-ticles are chemically modi�ed to develop the functional groups that can chemisorbenzymes. In the surface modi�cation reactions, the hydroxyl group can be used asa functional group on the particle surface. For instance, surface amino groups areintroduced on the silica surface by the reaction between the surface hydroxyl groupand 3-aminopropyltriethoxysilane (3-APTES). Indeed, in most methods, the surfaceof silica particles is chemically modi�ed only by the reaction of hydroxyl groups.

In addition to the hydroxyl groups, there exist siloxane bridges on the silicasurface. By heat treatment, the silica surface is easily dehydroxylated to formstrained siloxane bridges. They are reactive for reagents such as methanol.Nevertheless, siloxane bridges have not been applied as a functional site in thesurface modi�cation reactions for enzyme immobilization. By utilizing the reactivesiloxane as another functional site, the surface amino density is expected to becomehigher.

In this paper, surface modi�cation reactions and enzyme immobilization on silicaparticles are reported. A new reaction method is proposed to prepare the modi�edsilica particles for enzyme immobilization. The modi�cation process is carried outby a continuous �ow method, which can give high handling ef�ciency of the supportparticles. The fundamental properties of the surface reactions were investigated byusing a well-de�ned silica surface.

CSMP of silica particles for enzyme immobilization 233

2. SURFACE MODIFICATION REACTIONS

In the surface modi�cations, ethanolamine (2-aminoethanol) was used as the surfaceamination reaction agent. It is expected that the hydroxyl group of ethanolamineis reactive for both the hydroxyl groups and the reactive siloxanes on the silicasurface [1]. The reactions are similar to surface alkoxylation by alcohols [2]. Thesesurface amination reactions are expressed as:

SiOH C HOCH2CH2NH2¡H2O¡¡! SiOCH2CH2NH2; (1)

SiOSi C HOCH2CH2NH2 ¡! SiOCH2CH2NH2 C SiOH; (2)

where SiOH and SiOSi indicate the active surface hydroxyl group and reactivesiloxane, respectively. These reaction sites can be formed by preheating of theparticles [2–4]. SiOCH2CH2NH2 indicates the introduced surface amino groups.

In the usual surface modi�cation treatments, 3-APTES is applied as the aminationreagent. The surface amination reaction with 3-APTES proceeds only with theactive hydroxyl group [5]. On the other hand, the amination reaction withethanolamine proceeds on the reactive siloxanes as well as the active hydroxyls.By these reactions, it is possible to introduce densely packed amino groups on theparticle surface.

The surface aldehyde groups to immobilize enzymes are usually formed by thereaction with glutaraldehyde. However, because of the its volatility, glutaraldehydeis inappropriate as a vaporous reactant. Instead, glyoxal was used as the reagent forattaching the aldehyde group. The reaction between the amino group and glyoxal isexpressed as:

SiOC2H4NH2 C CHOCHO¡H2O¡¡! Si O C2H4N CH CHO: (3)

The surface aldehyde group acts as the chemisorption site for enzyme immobiliza-tion.

3. EXPERIMENTAL

The silica particles were Merck extra pure grade Silica gel 60. The average porediameter of this sample was about 12 nm [6]. Such a mesoporous silica gel is notnecessarily adequate to prepare highly active enzyme catalysts because of the masstransfer resistance for enzymes and reagents. However, since the surface propertieswere precisely determined, this sample was used in this fundamental study toanalyze the chemical reactions on the pure silica surface. The pretreatment of theparticles and the basic properties were previously reported [2–4, 6]. The number ofsurface hydroxyl groups and reactive siloxanes had been determined. Ethanolaminewas purchased from Kanto Kagaku (Japan) and glyoxal was purchased from TokyoKasei Kogyo (Japan).


Figure 1 shows a schematic view of the experimental apparatus for the priorsurface modi�cation reactions. Silica particles were preheated under a heliumor argon gas atmosphere. The gas velocity was set at 1.1 times the minimum�uidization velocity of the particle bed. The reactor tube was made of quartz glass.The inner diameter was about 20 mm and the gas inlet was about 1 mm. Thepreheating and the reaction temperatures were controlled by electric furnaces within§1±C. The gas line was heated by a �exible band heater to avoid condensation ofthe reagent vapors.

The treatments of the particles, from the preheating to the reaction with glyoxal,were carried out continuously in the apparatus shown in Fig. 1. We de�ned thisprocess as the continuous surface modi�cation process for enzyme immobilization(CSMP-EI). Since the heating and drying steps can be carried out without taking outthe particles, the handling of particles becomes much easier. The in�uence of thepreheating and the reaction temperature was investigated for the surface aminationreaction of the particles. The reaction with glyoxal vapor was carried out at roomtemperature.

Enzyme immobilization was examined on the particle surface modi�ed by CSMP.The adsorption amount of lysozyme was measured in order to examine the funda-mental properties of the particles. Lysozyme (obtained from Wako Pure ChemicalIndustries, Japan) was adsorbed from 0.05 M phosphate buffer solution. The con-centration of lysozyme was determined by spectroscopic analysis, using the biuretsolution as the color-producing agents. The absorbance at 510 nm was monitored

Figure 1. Experimental set-up for the CSMP-EI.


using a Nippon Bunko (Japan )V-530 type spectrometer. The total amount of ad-sorbed lysozyme was determined from the difference between the initial and �nalconcentrations.

The amount of chemisorbed (immobilized) enzyme was determined by thermalanalyses. In order to remove the physisorbed enzyme, the particles were washedwith the buffer solution, redistilled water and, �nally, ethanol. Each washing stepwas repeated several times, and then the particles were dried at room temperature ina vacuuming desiccator. Thermogravimetry (TG) and differential thermal analysis(DTA) analyses were carried out for the dried particles, using a Seiko Instruments(Japan) TG/DTA-320U. All thermal analyses were measured in air. The weightloss between 180 and 1200±C was measured for both the treated and the untreatedparticles. From the difference in the TG weight loss, the amount of chemisorbedenzyme was estimated. Also, infrared (IR) spectra were measured for the particlesurface by the diffusive re�ection method, employing a Nippon Bunko FT/IR-350with a DR-800/HS attachment. All spectra were measured under 10¡2 Pa with aresolution of 4 cm¡1.

The enzyme activity was tested for immobilized catalase on the particles preparedby the CSMP method. Catalase (from bovine liver) was obtained from Sigma-Aldrich (Japan). Hydrogen peroxide was decomposed by the immobilized catalase.The rate of the decomposition reaction was measured by a batch method in anErlenmeyer �ask under various conditions. A sample of 0.5 g of the preparedparticles or 0.25 mg of the material catalase was added as the catalyst into 100 dm3

of H2O2 solution. The concentration of H2O2 was determined by titration withKMnO4 solution. The enzyme activity was determined by correcting for blank testsusing untreated silica gel particles.

4. RESULTS AND DISCUSSION

4.1. Number of surface amino groups

The amination reaction was carried out under various experimental conditions. Pre-liminary experiments revealed that the optimum reaction temperature for ethanol-amine vapor was about 200±C. At higher temperatures, oxidation of ethanolamineproceeds and the number of formed surface amino groups decreases. Thus, the reac-tion temperature for ethanol amine vapor was set at 200±C through the experiments.

Figure 2 shows the number of surface amino groups NA formed by the reactionwith ethanolamine at 200±C for 2 h. NA was determined by TG analysis aspreviously reported [2, 4]. As the reaction time becomes longer than 90 min, thenumber of amino groups becomes nearly constant. These values show the maximumnumber of amino groups for each experimental condition.

It is seen that both the preheating temperature and the ethanolamine concentrationin�uence the number of surface amino groups. The increase in NA below 400±C canbe attributed to the increase in active hydroxyl groups formed by the preheating


Figure 2. Number of surface amino groups formed under various conditions.

treatment [2]. In this temperature range, the surface amino groups are formedmainly by (1) because of the low concentration of reactive siloxane bridges.

Above 400±C, the dependency of NA on the preheating temperature is affected bythe ethanolamine concentration. When the concentration is low, NA decreases withincreasing the preheating temperature. On the other hand, when the ethanolamineconcentration becomes higher (or contact with liquid ethanolamine by a Soxletextractor), NA increases with increasing the preheating temperature above 400±C.This is the same tendency as the case of surface alkoxylation with alcohols. Similarto the alkoxylation reaction, the increase of the surface amino groups in thistemperature range seems to be based on the reaction with the reactive siloxanebridges [2, 4]. In other words, (2) does not proceed when the concentration ofethanolamine is low.

Figure 3 shows the in�uence of the preheating temperature on the rate of thesurface amination reaction. It is evident that the rate of reaction increases withincreasing the preheating temperature. The reaction of the reactive siloxaneincreases the rate of the total surface reaction. The apparent rate constant basedon the total number of reaction sites can be estimated by applying the progressiveconversion model [3]. The results are listed in Table 1. The rate constant ofthe surface amination reaction kA is about the same value as that of the surfacealkoxylation by 1-butanol.

The preheating treatment of the silica particle above 400±C increases not only thesurface amino density, but also the rate of the amination reaction. The effect ofpreheating treatment on the amination reaction by ethanolamine can be understoodin the same way as alkoxylation with alcohols. The number of surface amino groupscan be controlled by the reaction conditions in the CSMP method.


Figure 3. Number of surface amino groups as a function of reaction time. Reaction temperature D200±C.

Table 1.Apparent rate constants for surface aminationwith ethanolamine

Preheating temperature .±/C kA (m3 /mol/ s)

400 3:64 £ 10¡5

600 5:02 £ 10¡5

From the experimental results for the surface amination and the reaction withglyoxal, the reaction conditions that gave the maximum surface amino density inCSMP were as follows: (i) preheating at 600±C for 2 h, (ii) surface amination at200±C for 1 h, (iii) drying at 200±C, (iv) reaction with glyoxal at 25±C for 2 h and(v) drying at 130±C. Using these treatments, the number of surface amino groupswas controlled to be 1.21 nm¡2, and about half of them were converted to aldehydegroups. This condition for the surface state means that about 30% of the originalsurface hydroxyl groups are modi�ed to amino groups [3]. Enzyme immobilizationwas tested on this surface state as an extreme case for the maximum surface aminodensity in CSMP. However, as the activity of the immobilized enzyme dependsstrongly upon the pre-modi�ed surface state (the densities of the surface aminoand aldehyde groups), this surface state does not necessarily provide the maximumenzyme activity.

4.2. Immobilization of lysozyme

Enzyme immobilization on the particles prepared by CSMP was examined, usinglysozyme as an example. Figure 4 shows the DTA pro�les of the particles at each


Figure 4. DTA pro�les of the particles at each stage in CSMP-EI.

stage in this process. The exothermic peaks reveal thermal decomposition of organiccompounds on the particle surface. The peaks become larger as the modi�cationproceeds and after immobilization of lysozyme, the peak intensity rapidly increases.Figure 5 indicates the IR spectra of the particle surface at each stage shown in Fig. 4.The change in absorption around 3000 cm¡1 indicates the progress of the surfacemodi�cation. Broad absorption is shown in the spectrum after immobilization oflysozyme. These facts indicate that enzymes can be chemically immobilized on thesilica surface modi�ed by CSMP.

Usually, glutaraldehyde is used as the modi�cation reagent to introduce the alde-hyde groups through the reaction with surface amino groups. When glutaraldehydewas used for the particles modi�ed to NA D 1:21 nm¡2, lysozyme was not immo-bilized on the particle surface. On the other hand, immobilization of lysozyme wascon�rmed for the particles modi�ed to NA D 0:58 nm¡2. These results indicate thatthe treatment with glutaraldehyde for the highly aminated surface is not effectivefor enzyme immobilization. Since the glutaraldehyde molecule has a longer chainthan glyoxal, it seems that the aldehyde groups at both ends could react with thedensely packed amino groups which were formed on the surface.

4.3. Adsorption of lysozyme

The adsorption of lysozyme was measured on the particles prepared by the CSMPmethod (modi�ed to NA D 1:21 nm¡2 and glyoxal vapor) under various conditions.


Figure 5. Infrared spectra of the particle surface at each stage in CSMP-EI.

It is well known that enzyme immobilization depends on the pH of the solution. Theamount of physisorbed lysozyme is also in�uenced by the solution pH [7–9]. Fromthe preliminary experiments for the CSMP particles, the amount of immobilizedlysozyme showed a maximum at pH 7.4. Thus, adsorption characteristics wereexamined at pH 7.4.

Figure 6 shows the dependence of adsorption on the adsorption time. The amountof physisorbed lysozyme 0p was determined as the difference between the totalamount of adsorption 0 and the amount of chemisorbed (immobilized) lysozyme 0c.The total adsorption increases in a few days and becomes almost constant aftercontact for 4 days. For mesoporous adsorbents such as silica gel used in this work,the rate of adsorption in solutions was much affected by the mass transfer rate inthe pores. In order to increase the immobilization rate, macroporous or non-poroussilica particles should be used as support materials.

Although the plateau in 0 indicates the apparent adsorption equilibrium, theadsorption state of lysozyme changes gradually. 0c increases with increasing contacttime, whereas 0p decreases gradually after the increase in the initial stage. Thisindicates that the ratio of chemisorbed lysozyme increases in the adsorbed layer,i.e. although the apparent equilibrium is reached in a few days, transition from thephysisorbed state to the chemisorbed state occurs in the adsorbed layer. However,


the higher-order structure may be destroyed over a long period and not all thechemisorbed molecules show the enzyme activity.

Figure 7 shows the adsorption isotherms measured after 48 h contact. Theadsorption amounts are plotted against the �nal concentration of the solution. Itis seen that 0c depends strongly on the concentration and the adsorbed layerformed at the higher concentration includes more chemisorbed molecules. Therepresentative immobilization yield was 40% at Cf D 3 mol/dm¡3. In order to

Figure 6. Dependence of adsorption amount of lysozyme on the contact time.

Figure 7. Amount of adsorbed lysozyme after contact for 2 days, plotted against the �nalconcentration.


increase the immobilization ef�ciency, the particles should be immersed in a higherconcentration solution.

4.4. Activity of prepared particles

Catalase was also easily immobilized on the particles prepared by the CSMPmethod. Figure 8 shows the IR spectra of the particles after contact with the

Figure 8. IR spectra of the particle surface after adsorption of catalase.


Figure 9. Decomposition of H2O2 catalyzed by immobilized catalase.

catalase solutions at various pH. The particles after contact with the solutionsbelow pH 7.4 show broad absorption bands around about 3000 cm¡1, whichindicate catalase immobilized on the particle surface. The optimum pH that gavethe most immobilized amount was 6.0. In order to compare the activity of theprepared particles, catalase was immobilized on the CSMP particles under thesame conditions (pH 6.0 and the initial concentration 6 g/dm3). The amount ofimmobilized catalase was estimated to be about 1% (0.01 g catalase / g particle) fromthe TG difference between the particles before and after the immobilization. Sincethe degree of surface modi�cation was easily controlled by the CSMP method, theamount of immobilized enzyme showed good reproducibility.

A representative decomposition curve of H2O2 catalyzed by the immobilizedcatalase is shown in Fig. 9, which indicates the enzyme activity of the preparedparticles. The rate of this decomposition reaction can be expressed as the �rst-orderreaction and the enzyme activity was estimated as the initial rate of reaction r0:

H2O2 .aq./ ¡! H2O.l/ C 1=2O2.g/; (4)

r0 D k0CH2O2 : (5)

The initial rate constant k0 at μ D 0 was determined by extrapolating the k –μ curve.Figure 10 shows the Lineweaver–Burk plots for the prepared particles and the

material catalase. It is seen that a linear relation holds also for the immobilizedcatalase. This indicates that the apparent rate of reaction can be expressed by theMichaelis– Menten equation:

r0 D rmaxCH2O2

Km C CH2O2

; (6)


Figure 10. Lineweaver–Burk plot for decomposition of H2O2.

Table 2.Km and speci�c activity for decomposition of H2O2 at 1±C (pH D 7:0)

Km (mol/dm3) Speci�c activity (U/mg)

Material catalase 0.055 7000Immobilized catalase 0.171 350

where r0 and CH2O2 are the initial rate and the initial concentration, respectively. TheMichaelis constant Km and the speci�c activity calculated from the maximum ratermax are listed in Table 2. These kinetic parameters for the immobilized catalaseare apparent values which include the in�uence of the mass transfer resistance inthe particles. Although the speci�c activity decreases with immobilization, it iscon�rmed that the particles prepared by the CSMP method possess a certain enzymeactivity.

The activity of immobilized catalase in this work was much lower than the freeenzyme. The decrease in activity is attributed to two factors. One is the masstransfer resistance in pores of support particle. Since the support particle in thiswork was mesoporous silica gel, the overall reaction rate in solutions was controlledby the diffusion in pores. For practical use of enzyme catalysts, macroporousor non-porous particles should be used as support materials. The CSMP methodcan also be applied to such particles. Another factor for decreasing the activityis deactivation of enzyme by immobilization. On the highly aminated surface,the enzymes are immobilized by forming multi-chemical bonds. In this work, ascatalase was immobilized on the surface of the maximum surface amino densityby CSMP, the activity was decreased by the change in the higher-order structure.


Figure 11. Stability of catalase in aqueous solutions. Material and immobilized catalase were soakedin phosphate solutions (pH 7) at 5±C.

For preparation of the actual enzyme catalysts, more studies are needed as to therelationship between the surface amino density and the enzyme activity.

The stability of the immobilized catalase was examined by soaking the particlesin water at pH 7.0 and 5±C. The decrease in the enzyme activity was evaluated asthe decrease in the initial rate of reaction. Figure 11 shows the decrease in theactivity as a function of storage time. The material and immobilized catalase wereinactivated gradually in water and the half-life was observed to be approximately110 and 140 h, respectively. The immobilized catalase was a little more stable thanthe material, especially in the initial stage.

5. CONCLUSIONS

The surface modi�cation process examined in this work is useful for immobilizationof enzymes on silica particles. The surface modi�cation reactions can be carriedout continuously and the handling ef�ciency of the particles becomes higher. Byusing an amino alcohol (ethanolamine in this work) as the modi�cation reagent, thesurface of silica particles is effectively aminated. Both the surface hydroxyl groupand the reactive siloxane on silica are available for the surface amination reaction ofthe particles. The number of surface amino groups can be controlled by the reactionconditions. The surface-modi�ed particles for enzyme immobilization are easilyprepared in this CSMP method with high handling ef�ciency of the particles, whichmakes it easier to prepare enzyme catalyst particles on a large scale.

Enzymes can be immobilized on the particles prepared by the CSMP method.The amount of immobilized enzyme was controlled by the degree of modi�cationas well as the enzyme concentration. In order to control the degree of surface


modi�cation, the CSMP method is a useful method that gives good reproducibilityfor the chemical state of the surface.

Enzyme activity was con�rmed for immobilized catalase prepared by the CSMPmethod. The activity of the immobilized enzyme in this work was much lower thanthe free enzyme because of the mass transfer resistance in pores and the change inhigher-order structure of the enzyme. More active catalysts could be prepared frommacroporous or non-porous particles as support materials by the CSMP method.Also, the CSMP method could be applied to ceramic particles other than silica. Thisprocess is expected to be an effective method to prepare various enzyme catalysts.

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continuous surface modification of silica particles for enzyme immobilization

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