Biochemical Engineering Journal 8 (2001) 171–174

Short communication

Preparation of immobilized enzyme gel particles using anelectrostatic atomization technique

Hideo Watanabe∗, Tatsushi Matsuyama, Hideo YamamotoDepartment of Bioengineering, Faculty of Engineering, Soka University, Hachioji, 192-8577 Tokyo, Japan

Received 30 May 2000; accepted 16 March 2001

Abstract

To prepare small size particles of immobilized enzyme with high catalytic effectiveness factor, an electrostatic atomization technique wasapplied. Sodium-alginate solution containing enzyme was dispersed dropwise into CaCl2 aqueous solution by applying dc high voltage toprepare gel beads. The catalytic performance of the prepared immobilized-enzyme particles is described with evaluating the effectivenessfactor.

The prepared beads had the minimum diameter of 100 �m, which was one order smaller than that attainable by conventional droppingmethods. The diameter can be controlled by changing the operating conditions, such as applied voltage and volumetric flow rate ofthe solution. The immobilized enzyme particles had higher effectiveness factors compared to those prepared by conventional methods.© 2001 Elsevier Science B.V. All rights reserved.

Keywords: Electrostatic atomization; Immobilized enzyme; Calcium-alginate gel; Heterogeneous biocatalysis; Kinetic parameters

1. Introduction

Usually alginate gel beads, which are often used as amatrix for immobilization of biocatalysts, are prepared bydropping method. Sodium-alginate solution is poured drop-wise into a gelling bath through a capillary tip [1]. Thismethod gives beads diameter of millimeter order. The useof gel beads of this size to immobilize enzyme results ina demerit that the reaction rate is limited by diffusion ofsubstrates and products inside the particles. One of thesimplest methods to increase the effectiveness factor is toapply smaller gel (catalysts) beads.

In the present work, we propose a method, i.e. electro-static atomization technique to prepare alginate gel beads.Namely, an aqueous sodium-alginate solution containingenzymes was dispersed into a CaCl2 aqueous solution assmall droplets by applying dc high voltage. The electrostaticatomization is a well known phenomenon where liquid isdispersed into fine droplets by an electrostatic force work-ing on the surface of the liquid. This technique has beenapplied to many different fields, e.g. ink-jet printers, paintsprayers and crop sprayers [2–4].

∗ Corresponding author. Tel.: +81-426-91-9454; fax: +81-426-91-9454.E-mail address: hwatanab@edu.t.soka.ac.jp (H. Watanabe).

In previous studies [5,6], although other atomization tech-niques were presented, catalytic activity of the immobilizedbiocatalyst was not discussed in detail. Accordingly, thispaper discusses the catalytic performance and efficiency ofthe immobilized enzyme obtained by the electrostatic atom-ization and evaluated in terms of the catalytic effectivenessfactor.

2. Experimental

2.1. Materials

Sodium-alginate (Kanto Kagaku) as an immobilizing ma-trix, calcium chloride (Wako pure chemical industries) as agelling agent, sucrose (Wako) as the substrate of enzyme re-action, and invertase (Beehringer–Mannhein) as the enzymeto be immobilized, were purchased. They were used withoutfurther purification. Glucoseoxidase, peroxidase and ABTS(2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) asthe indicator agent (Beehringer–Mannhein), were used forthe assay of glucose produced from the invertase reaction.

2.2. The electrostatic atomization

Fig. 1 shows the experimental apparatus for electrostaticatomization. Sodium-alginate solution 10–30 g/l containing

1369-703X/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S1369 -703X(01 )00121 -8

172 H. Watanabe et al. / Biochemical Engineering Journal 8 (2001) 171–174

Nomenclature

Ce concentration of enzyme in the gel (kg/m3)Cs concentration of substrate in reaction

medium (mol/m3)Csr concentration of substrate in internal

surface of gel (=CsK) (mol/m3)Dse effective diffusion coefficient of the

substrate in the gel (m2/s)kmax maximum reaction rate constant in

Mechaelis–Menten reaction (mol/kg s)K partition coefficientKm Michaelis constant (mol/m3)R radius of the immobilized enzyme

particle (m)vs reaction rate in the reactor (mol/m3 s)V volume of reaction medium (m3)Vse reaction rate inside the immobilized

enzyme (mol/m3 s)Vsr reaction rate with no diffusion

limit (mol/m3 s)w weight of the immobilized enzyme

particle (kg)Y enzyme immobilization yieldφm Thiele modulusη effectiveness factorρ density of the immobilized enzyme

particle (kg/m3)

enzyme with 1.0 g/l in case of enzyme immobilization, wasflown out from a nozzle electrode (∅ 6 mm × 2 mm brasstube, tip o.d. = 0.51 mm, i.d. = 0.20 mm) and dc highvoltages from 0 to 5 kV were applied between nozzle andearth electrodes (3 mm brass plate, 51 mm × 69 mm, with a

Fig. 1. Schematic diagram of experimental apparatus for electrostaticatomization.

hole ∅ = 10 mm at the center of the plate). The dropletsof the solution were dripped into 5–20 g/l calcium chloride(CaCl2) aqueous solution. The diameter of the Ca-alginatebeads was measured under a microscopic observation. Theviscosity of sodium-alginate solution measured using of arotational viscometer (Vismetron VS-A1, Shibaura Systems)is 1.23 × 10−1, 1.09, 4.00 Pa s for the concentrations of 10,20 and 30 g/l, respectively.

2.3. Enzyme immobilization yield

During the process of the enzyme immobilization, the en-zyme was released partly to CaCl2 solution and not immo-bilized. The amount of the enzyme released was measuredafter immobilization. The amount of the enzyme containedin Ca-alginate gel beads was determined from the differencebetween the amounts in the CaCl2 solution and those in theoriginal solution. The ratio of the amount of the enzyme inthe gel to that in original solution was defined as “enzymeimmobilization yield”.

2.4. Assay of immobilized enzyme

After the gelation in CaCl2 solution, particles immobi-lizing enzyme were incubated at 277 K for 24 h to stabilizethe gel matrices and then the particles were filtered witha sheet of nitrocellulose membrane (1.0 �m pore size). Apart of the particles was used for reaction experiments. En-zyme reaction was carried out at 313 K and pH 4.6 with35 mmol/l sucrose solution stirred vigorously by a magneticstirrer (rotation speed 400 rpm). Since, invertase catalyzesthe hydrolysis of sucrose into glucose and fructose, amountsof glucose from the enzyme reaction were measured in timecourse using glucoseoxidase–peroxidase and ABTS [7].

2.5. Heterogeneous catalytic effectiveness factor

The catalytic performance of immobilized enzymes cangenerally be discussed by catalytic effectiveness factor η.From the definition [8], η is expressed as

η = Vse

Vsr, (1)

where

Vse = vs

w/ρV, (2)

and

Vsr = kmaxCeCsr

Km + Csr. (3)

The effectiveness factor is a function of Thiele modulus [8]φm,

φm = R

√kmaxCe

KmDse. (4)

H. Watanabe et al. / Biochemical Engineering Journal 8 (2001) 171–174 173

Km and kmax of free invertase were experimentally obtainedas 35.1 mol/m3 and 1.23 × 10−2 mol/(g s), respectively. Dseand K of sucrose in Ca-alginate gel beads were measuredby monitoring a non-steady state diffusion process fromthe gel particle to external solution [9]. The values of Dsewere 4.5, 3.9, 3.6, 4.2 × 10−10 m2/s at a concentration ofalginate/CaCl2 of the original solution 10/20, 20/20, 30/20,20/5 g/l, respectively. The values of K were 0.97 on eachbead and were not sensitive to the concentration under theexperimental conditions.

3. Results and discussion

3.1. Electrostatic atomization of sodium-alginate solution

Fig. 2 shows the dependency of the Ca-alginate beadsdiameter on the applied voltage. The mean diameter onthe vertical axis represents average value over 50 beads.The relative standard deviations of the diameters were not>10%. The critical voltage, from electrostatic dropping toelectrostatic atomization, Vc was about 3.5 kV. The diame-ter of Ca-alginate beads about 2 mm at 0 kV, and decreasedwith the increase of the applied voltage and finally reached200–300 �m on 5.0 kV. From the figure, the decrease in thediameter was not so sensitive to the applied voltages ex-ceeding Vc. It was also found that the diameter was directlyproportional to a power of the volumetric flow rate. Thus,applied voltage and volumetric flow rate were changed toprepare immobilized enzyme particles on <1 mm in diam-eter. A more detailed study on the electrostatic atomizationof highly viscous liquids will be presented elsewhere.

3.2. Enzyme immobilization yield

Fig. 3 shows the relationship between enzyme immobi-lization yield Y and the diameter of the immobilized enzyme.

Fig. 2. Dependency of diameter of Ca-alginate beads on appliedvoltage, where distance of electrodes = 5 mm and volumetric flowrate = 1.33 × 10−2 ml/min.

Fig. 3. Relationship between enzyme immobilization yield and diameterof immobilized enzyme particle: plots, experimental data; lines, regressioncurves.

Y decreased with the decrease in diameter, which makes toassume that the amount of enzyme escaped into CaCl2 solu-tion during the immobilization is directly proportional to thespecific surface area of the beads. The curves in Fig. 3 weredrawn on the basis of this assumption, and their likeness tothe data showed a good account. From the comparison ofthe keys in Fig. 3, it can be stated that the immobilizationyield increased at high concentration of the alginate and/orCaCl2 solution. The following reasons are considered. Thegelation rate increased owing to the enhancement of theion-exchange rate at the gelling process with the increase ofthe concentration of Ca2+. The diffusion rate of the enzymedecreased due to the rise in the alginate molecular densityin the process caused by the increase in the concentrationof the alginate. Accordingly, applying a higher concentra-tion of alginate and CaCl2 had an advantage regarding theenzyme immobilization yield, but may have disadvantageregarding the catalytic activity of the immobilized enzymebecause of their low Dse.

3.3. Performance of the immobilized enzyme

Fig. 4 shows the relationship between η and the diameterof the immobilized enzyme. The theoretical lines calculatedfrom the Thiele modulus [8] were also shown. The valuesof η for smaller particles <1 mm in diameter prepared bythis method are higher compared with those of larger onesprepared without applying electric field. The increasingdependency of the effectiveness factor with the decreasein diameter agreed with that of the theoretical values forall conditions, while the experimental values were smallerthan the theoretical ones. The ratios of experimental valuesto theoretical ones were independent of the diameter andthe value was constant at 0.35. This value may be attributedto the change in kinetic parameters of the enzyme (such askmax and Km) influenced by the immobilization process.

174 H. Watanabe et al. / Biochemical Engineering Journal 8 (2001) 171–174

Fig. 4. Relationship between effectiveness factor and diameter of immobi-lized enzyme particle: plots, experimental values; lines, theoretical values.

Fig. 5. Plots of ηY against diameter of immobilized enzyme particle.

Fig. 5 shows the relationship between ηY and the di-ameter of the immobilized enzyme particle. ηY indicatesthe efficiency of the enzyme activity based on the initialamount of the enzyme applied to the immobilization. ηYwas found to have the optimum value at the immobilization

conditions, 30 g/l sodium-alginate, 5.0 kV in applied voltageand 1.33 × 10−2 ml/min in volumetric flow rate. However,if the enzyme exists a priori in CaCl2 solution, the enzymerelease can be prevented during the immobilization process.In such cases, higher efficiency can be expected.

4. Conclusions

In order to prepare immobilized enzyme particles of smallsize, the electrostatic atomization technique was applied.Ca-alginate beads with small diameter, down to 100 �m,could be prepared. The size is one order smaller than thatproduced without applying electric field, and diameter couldbe controlled using applied voltage and volume flow rate asoperating variables.

The enzyme immobilization yield decreased with the de-crease in diameter of the particles because of the increasein the specific surface area. Concerning the efficiency basedon the amount of input enzyme, there was the optimumimmobilization condition, 30 g/l sodium-alginate, 5.0 kV inapplied voltage and 1.33 × 10−2 ml/min in volumetric flowrate for the range of experiments. This method was usefulfor improving the performance of immobilized enzymeparticles by giving them less diameter.

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