Biotechnol. Bioprocess Eng. 2001, 6: 150-155
`
`Effects of Water and Silica Gel on Enzyme Agglomeration in
`Organic Solvents
`Keehoon Won and Sun Bok Lee*
`
`Department of Chemical Engineering, Division of Molecular and Life Sciences, and Institute of Environmental
`and Energy Technology, Pohang University of Science and Technology, San 31, Hyoja-Dong, Pohang 790-784, Korea
`
`Abstract It has been observed that water, which is absolutely essential for enzyme activity,
`can induce the agglomeration of enzyme particles in organic media. Although enzyme ag-
`glomeration is significant in that it usually reduces enzyme activity and stability, little atten-
`tion has been paid to the quantitative analysis of enzyme agglomeration behavior in nona-
`queous biocatalytic systems. In this study, the effects of water and silica gel on enzyme ag-
`glomeration were investigated using Candida rugosa lipase and cyclohexane as a model en-
`zyme and an organic medium. The extent of enzyme agglomeration was quantified by sieve
`analysis of freeze-dried agglomerates. Increasing the water content of the medium increased
`the size of the enzyme agglomerates, and it was found that water produced during the esteri-
`fication reaction could also promote the agglomeration of enzyme particles suspended in or-
`ganic media. On the other hand, the size of the enzyme agglomerates was remarkably re-
`duced in the presence of silica gel at the same water content. We also show that this increase
`in the size of enzyme agglomerates results in lower reaction rates in organic solvents.
`
`Keywords: Candida rugosa lipase, enzyme agglomeration, sieve analysis, silica gel, water con-
`tent
`
`INTRODUCTION
`
`In recent years, the use of enzymes in nonaqueous
`solvents has been extended and the technique has
`found a variety of applications [1,2], which is due to
`several advantages, including, increased solubility of a
`hydrophobic substrate, shift of an equilibrium in a de-
`sired direction, and the possibility of conducting reac-
`tions that are impossible in water [3]. Although organic
`solvents in place of water are used as the reaction media,
`it has been recognized for many years that water is ab-
`solutely essential for enzymatic catalysis. Indeed water
`not only participates in all non-covalent interactions,
`which maintain protein conformations, but also plays a
`crucial role in enzyme dynamics. Therefore, it is gener-
`ally accepted that water is essential for enzymes in or-
`ganic solvents and that the hydration level of an en-
`zyme significantly affects its catalytic activity. However,
`an increase in the hydration level of enzymes is not al-
`ways accompanied by an increased enzymatic activity
`in microaqueous reaction systems. In general, if there is
`too little water, the reaction rate will be low or zero,
`because of a loss of catalytic activity of the enzymes,
`and if there is too much, then other undesirable effects
`of the excess water may lower the reaction rates. Con-
`sequently, in most cases there exists an optimal water
`
`* Corresponding author
`Tel: +82-54-279-2268 Fax: +82-54-279-2699
`e-mail: sblee@postech.ac.kr
`
`content. It is expected that the rate of enzyme reactions
`in organic solvents can be raised if adverse effects due to
`excess water are diminished. It has been suggested that
`the excess water has unfavorable effects, for example, it
`may cause hydrolytic reverse reactions, the partitioning
`of substrates, enzyme inactivation, or the agglomera-
`tion of enzyme particles.
`In our previous study, we demonstrated that enzyme
`agglomeration was probably the most significant of the
`aforementioned factors [4]. It is believed that enzyme
`agglomeration as induced by water in organic solvents
`is purely a physical phenomenon, which is also known
`as spherical agglomeration. When dried barium sulphate
`is agitated with dry benzene, spherical agglomerates ca.
`0.5-1.0 mm in diameter are formed [5]. Moreover, fine
`particles dispersed in a liquid suspension can be ag-
`glomerated by adding a small amount of a second im-
`miscible bridging liquid, which preferentially wets the
`particles [6-10]. A significant number of studies have
`been conducted on the spherical agglomeration of a va-
`riety of chemical and pharmaceutical powders. Enzyme
`agglomeration in microaqueous media has also been
`observed by many workers [11-13], but little attention
`has been paid to quantifying the effect. Previously, only
`one research group [14] has reported measuring enzyme
`particle sizes in organic solvents by direct microscopy
`using a graduated eyepiece and investigated the effects
`of hydration on the size of enzyme powder dispersions.
`However, there has been no report on the effect of addi-
`tives such as silica gel on enzyme agglomeration. In the
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`Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 2
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`present work, we investigated the water-induced ag-
`glomeration of enzyme particles in organic solvents,
`particularly with respect to the effect of silica gel on
`sizes of enzyme agglomerate. Enzyme agglomerates
`were quantified by sieve analysis. We also examined the
`effect of enzyme agglomerate size on the rate of enzy-
`matic reactions in organic solvents.
`
`MATERIALS AND METHODS
`
`Enzyme and Reagents
`
`Lipase OF from Candida rugosa was purchased from
`Meito Sangyo (Tokyo, Japan) and used as received. Sil-
`ica gel (product number: S0507, particle size: 40-63 µm)
`was from Sigma (St. Louis, MO, USA). Octanoic acid
`and butanol were also from Sigma (St. Louis, MO, USA).
`Cyclohexane was obtained from J. T. Baker (Phillipsburg,
`NJ, USA). All chemicals used in this work were of ana-
`lytical grade and were used without further purification.
`Enzyme powders were equilibrated with saturated salt
`solution (LiCl) prior to use.
`
`Esterification Reaction in Organic Solvents
`
`Unless otherwise specified, esterification reactions
`were performed in the following manner. Lipase OF (1.2
`g) with or without silica gel (2.4 g) was suspended in
`cyclohexane solution containing 0.2 M of octanoic acid.
`This suspension was sonicated to disperse enzyme par-
`ticles. Another mixture composed of water added and
`cyclohexane was prepared and also sonicated. The two
`prepared stocks were mixed together and then incu-
`bated with stirring at 30ºC and 250 rpm for 10 min.
`The reactions were carried out in a total volume of 120
`mL in a 250 mL spinner flask with an over-head impel-
`ler (Bellco Glass Inc., USA) at 30ºC and 250 rpm. The
`reaction was initiated by adding 0.4 M of butanol. At
`predetermined intervals, 120 µL samples were with-
`drawn and subjected to GC analysis.
`
`Quantification of Enzyme Agglomeration
`
`Powdered lipase OF weighing 1.2 g with or without
`2.4 g of silica gel was put into a 250 mL spinner flask
`with an over-head impeller and 70 mL of cyclohexane
`solution containing 0.2 M of octanoic acid was added.
`In another vessel, water was added to 50 mL of cyclo-
`hexane. The above preparations were sonicated sepa-
`rately and then mixed together. This suspension was
`agitated at 30ºC and 250 rpm for 10 min, and then the
`resulting agglomerates were collected in freeze-drying
`flasks and were freeze-dried overnight. No significant
`changes in agglomerate size after freeze-drying were
`observed. The freeze-dried enzyme agglomerates were
`first analyzed photographically. The size distribution of
`the enzyme agglomerates was determined by sieve
`analysis using standard sieves (Chung Gye Industrial
`Manufacturing Co., Korea). The size of sieves used were
`
`151
`
`as follows: 0.038, 0.053, 0.063, 0.106, 0.250, 0.355, 0.50,
`0.60, 0.71, 0.85, 1.0, 1.18, 1.40, 1.70, 2.0, 2.36, 2.80, 3.35,
`4.0, 4.75, and 5.6 mm. Sieve analysis was performed by
`stacking the sieves in ascending sieve size order and
`placing the enzyme agglomerates in the top sieve. A
`closed pan, a receiver, was placed at the bottom of the
`stack to collect the fines and a lid was placed at the top
`to prevent powder loss. The stack was shaken at 150
`rpm for 10 min and the residual weight of the agglom-
`erates on each sieve was determined. Results are ex-
`pressed as fractional weight percentage retained. The
`weight mean sphere diameter for each run was calcu-
`lated from the size distribution [6].
`
`Analysis of Substrates and Product
`
`The concentrations of octanoic acid and butyl octa-
`noate were analyzed by gas chromatography [Hewlett
`Packard 5890 Series II (USA)] using a cross-linked poly-
`ethylene glycol capillary column (HP-INNOWax, 30 m
`× 0.32 mm). Helium was supplied as a carrier gas at a
`rate of 2 mL/min. Hydrogen and air were supplied to
`the FID at 33.4 mL/min and 330 mL/min, respectively.
`The injector and the FID temperatures were 250ºC and
`275ºC respectively. The oven temperature was pro-
`grammed for 2 min at 170ºC and was increased to
`240ºC at a rate of 20ºC/min and then maintained at
`240ºC for 1 min. One µL samples were injected.
`
`RESULTS AND DISCUSSION
`
`Effect of Water Contents on Enzyme
`Agglomerate Size
`
`Effect of water content on enzyme agglomerate size
`was investigated by adding different amounts of water
`to the organic solvent. The amount of water added ex-
`ceeded the solubility limit (<0.01% v/v) and therefore,
`because no discrete water phase was observed, the ex-
`cess water must have associated with the enzyme par-
`ticles. The images shown in Fig. 1 are of enzyme ag-
`glomerates, which were photographed after freeze dry-
`ing. Fig. 1 shows that the shapes of the enzyme ag-
`glomerates are non-uniform and that their sizes in-
`crease with increasing amounts of water added. A small
`amount of water added to the organic solvent plays a
`role as a bridging liquid. Although the mechanism of
`spherical agglomeration has not yet been fully eluci-
`dated, it has been proposed that the bridging liquid ex-
`erts a marked influence on agglomeration [10]. Gener-
`ally the size of the spherical agglomerates increase with
`increasing levels of bridging liquid [8]. The size distribu-
`tions of the enzyme agglomerates obtained from sieve
`analysis are shown in Fig. 1. The weight mean sphere
`diameter was 1.4 mm with 0.6 mL of water added
`(0.5% v/v), and 3.2 mm when 1.2 mL of water was
`added (1.0% v/v). The weight mean sphere diameter of
`enzyme agglomerates increased by more than a factor
`of two. Enzyme agglomeration was also examined on
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`Fig. 2. Photographs and size distribution of the enzyme ag-
`glomerates induced by esterification without silica gel. (a)
`Before the reaction and (b) after 5 h reaction (conversion =
`100%). The esterification reaction was carried out at 30ºC and
`250 rpm without silica gel.
`
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`addition. During esterification water is produced as a
`by-product. Much of the water produced during the
`reaction is adsorbed by the enzymes. Because water can
`cause enzyme particles dispersed in organic medium to
`agglomerate, it might be expected that the enzyme par-
`ticles would be larger after reaction. The photographs of
`enzyme agglomerates before and after reaction are
`shown in Fig. 2. When the reaction time was 5 h, con-
`version was 100 % and 0.2 M of water (0.432 mL) was
`formed. Fig. 2 shows that the weight mean sphere di-
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`Fig. 1. Photographs and size distribution of the enzyme ag-
`glomerates induced by adding water in the absence of silica
`gel. The amount of water added: (a) 0.6 mL; (b) 1.2 mL. The
`enzyme agglomeration occurred under the conditions used for
`the esterification reaction i.e., 30ºC and 250 rpm for 10 min
`and then the resultant enzyme agglomerates were freeze-dried
`overnight.
`
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`adding more water. However, water additions of greater
`than 1.8 mL, to the medium, resulted in the solid mate-
`rials adhering to the flask walls and bottom, and there-
`fore, this beyond the scope of our experiment. Further
`water addition lead to a separate water which con-
`tained dissolved enzyme.
`For further investigations, enzymatic esterification in
`organic solvents was carried out without further water
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`Fig. 3. Photographs and size distribution of the enzyme-silica gel agglomerates induced by adding water in the presence of silica gel.
`The amounts of water added were: (a) 0.6 mL; (b) 1.2 mL; (c) 1.8 mL; (d) 2.4 mL. Enzyme agglomeration occurred at 30ºC and 250
`rpm for 10 min and the resultant enzyme agglomerates were freeze-dried overnight.
`
`ameter of the enzyme before the reaction was 0.56 mm,
`and after the reaction was 1.3 mm. This value is consis-
`tent with a weight mean sphere diameter of 1.4 mm at
`0.6 mL of added water. This result, when viewed in con-
`junction with the previous result, suggests that the wa-
`ter produced by the reaction within the medium can
`also agglomerate suspended enzyme particles.
`
`Effect of Silica Gel on Enzyme Agglomerate Size
`
`The effect of silica gel on enzyme agglomerate size
`
`was analyzed by adding various amounts of water to
`the organic medium. Silica gel was simply added to the
`medium before agglomeration took place. The pictures
`shown in Fig. 3 are of enzyme-silica gel agglomerates,
`which were freeze-dried after agglomeration. As shown
`earlier, the agglomerate size was found to increase
`gradually as the amount of added water increased. The
`size distributions of these agglomerates are also shown
`in Fig. 3. The size distribution curves show two differ-
`ent peaks. The first sharp peak that appeared at about
`100 µm was mainly due to the presence of silica gel, and
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`Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 2
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`Fig. 5. Effect of the size of enzyme agglomerates on reaction
`rate. The size of used enzyme agglomerates: (!) < 1.0 mm;
`(") 1.0-2.0 mm; (!) 2.0-3.35 mm.
`
`observed without silica gel at the same water content
`(Fig. 1(a) and (b)), which is explained by the partition-
`ing of water to the silica gel. Hydrophilic silica gel as
`well as enzyme particles in organic medium can adsorb
`water [15]. Hence, in the presence of silica gel, the
`amount of water available to the enzyme particles is
`reduced, and this results in a reduction of enzyme ag-
`glomerate size.
`In addition, enzyme agglomeration was observed dur-
`ing the esterification reaction in the presence of silica
`gel. The reaction conditions used were the same as
`those described in Fig. 2. In common with the results
`obtained without silica gel, conversion reacting for 5 h
`was 100%, and 0.2 M of water (0.432 mL) was pro-
`duced. Photographs and size distributions of agglomer-
`ates before and after this reaction are shown in Fig. 4.
`In contrast to the results obtained in the absence of
`silica gel, the weight mean sphere diameter varied only
`slightly in the presence of silica gel (from 0.55 mm to
`0.51 mm). The reason for this may be that silica gel
`added to the reaction medium adsorbs much of the wa-
`ter produced during the course of the reaction and
`therefore, the amount of water around enzyme parti-
`cles remains almost constant. Other investigators have
`also used silica particles to buffer the water content
`during a reaction [15-17].
`
`Effect of Enzyme Agglomerate Size on the
`Reaction Rates
`
`In our previous study, it was suggested that enzyme
`agglomeration should be considered to be the most sig-
`nificant cause of reduced enzymatic activity at high
`water [4], and it has been shown in this study that en-
`zyme particles suspended in organic media are agglom-
`erated by water and that the agglomerate size increases
`with increasing amounts of water. Lyophilized enzyme
`agglomerates were divided into three groups according
`to their sizes, and enzymatic esterification was per-
`formed using three enzyme preparations in cyclohexane.
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`Fig. 4. Photographs and size distributions of the enzyme ag-
`glomerates induced by the esterification with silica gel. (a)
`Before the reaction and (b) after 5 h reaction (conversion =
`100%). The esterification reaction was carried out at 30ºC and
`250 rpm with silica gel.
`
`hence, excluded from the calculation of weight mean
`sphere diameter of the enzyme agglomerates. The
`weight mean sphere diameters calculated in this man-
`ner were 0.84, 1.4, 1.7, and 2.0 mm at 0.6, 1.2, 1.8, and
`2.4 mL of added water, respectively.
` In the presence of silica gel, the size of the enzyme
`agglomerates also increased upon adding water to the
`medium. However, as Fig. 3(a) and (b) show, the size of
`the enzyme agglomerates was much smaller than that
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`The effect of enzyme agglomerate size on reaction rates
`is shown in Fig. 5. In all cases, the time-course of the
`reaction showed a sigmoidal shape because of an initial
`insufficiency of water essential for enzymatic catalysis.
`A clear dependence was found between the determined
`reaction rates and the size of the enzyme agglomerates.
`Specifically, as the size of the enzyme agglomerates in-
`creased, the reaction rates decreased, which can be ex-
`plained in terms of mass transfer limitations. In general,
`enzymes are insoluble in organic solvents but can oper-
`ate work in a suspended state. Unlike solubilized en-
`zymes in an aqueous solution, enzymes which catalyze
`reactions in organic solvents are heterogeneous cata-
`lysts, and thus are subject to diffusional limitations. It
`is well known that reactions catalyzed by enzyme par-
`ticles suspended in nonaqueous solvents are diffusion-
`ally limited [18]. Mass transfer limitations are depend-
`ent upon a number of factors, which include the size of
`the particle. Kamat et al. [19] showed that enzyme par-
`ticles suspended in anhydrous organic solvents would
`be subject to increasing diffusional limitations as the
`enzyme particle size increased. Thus the size increase
`due to enzyme agglomeration can cause reduced reac-
`tion rates.
`
`CONCLUSION
`
`Water-induced enzyme agglomeration, which can de-
`crease enzyme activity and stability in organic solvents,
`was investigated and quantified by sieve analysis. It
`was shown that water increased the size of the enzyme
`agglomerates, which resulted in the reduction of reac-
`tion rates. In addition, it was demonstrated that a sim-
`ple addition of silica gel to the reaction media could
`effectively prevent enzyme agglomeration, a serious
`problem for enzyme reactions in nonaqueous media. In
`this work, enzyme agglomeration was quantitatively
`analyzed for the first time and the experimental results
`obtained show that enzyme agglomerate size is one of
`key factors that should be taken into account when
`considering enzymatic reactions in organic solvents.
`
`Acknowledgements This study was supported by
`Korean Ministry of Education through Research Fund.
`
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`[Received March 30, 2001; accepted April 24, 2001]
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