Activity of Enzymes Immobilized in Colloidal Spherical Polyelectrolyte

Biomacromolecules 2005, 6, 948-955

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Activity of Enzymes Immobilized in Colloidal Spherical Polyelectrolyte Brushes B. Haupt, Th. Neumann, A. Wittemann, and M. Ballauff* Physikalische Chemie I, Universita¨t Bayreuth, Universitaetsstrasse 30, D-95440 Bayreuth, Germany Received October 12, 2004; Revised Manuscript Received December 7, 2004

We investigate the enzymatic activity of glucoamylase and β-glucosidase adsorbed on a novel type of colloidal particles. The particles used consist of a poly(styrene) core onto which long chains of poly(acrylic acid) or of poly(styrene sulfonic acid) are grafted (“spherical polyelectrolyte brush”). Proteins adsorb spontaneously onto these particles from aqueous solutions if the ionic strength is low. Moreover, the colloidal stability is not impeded by the adsorbed proteins despite the fact that up to 600 mg of enzyme is adsorbed per gram of the carrier particles. The activity of immobilized glucoamylase and β-glucosidase adsorbed onto these particles is analyzed in terms of the Michaelis-Menten parameters. This analysis shows that both enzymes keep nearly their full activity. The Michaelis constant KM differs only slightly from the KM value of the native enzyme when the amount of adsorbed enzyme is raised despite the high local concentration of immobilized enzymes. All data demonstrate that spherical polyelectrolyte brushes present a novel way to immobilize enzymes. Introduction Immobilization of enzymes on solid supports is an important problem of biotechnology.1-3 Up to now, a great variety of systems designed for the purpose have been introduced and discussed. Supports suitable for technical applications should maintain a high level of enzyme activity while preventing a possible leaching out during the reaction.2 Colloidal particles are well-suited for the immobilization of enzymes because of the large surface created in suspensions of objects of a few 100 nm in diameter.4 The immobilization of the biomolecules on colloidal particles can be done by adsorption from solution, and a great number of studies devoted to this problem is available.1,5-16 Adsorption onto solid surfaces may lead to a deformation of the enzyme that may be partially reversible upon desorption.13,16 The flattening and deformation of adsorbed proteins is well documented for planar macroscopic surfaces17-23 and for colloidal particles.6,20 Immobilization on colloidal particles can also be done by building up polyelectrolyte multilayers on the surface of the particles.24-29 Up to now, only a few studies devoted to a quantitative analysis of the activity of enzymes bound to colloidal particles are available.15,24,25,28,29 Application of the classical Michaelis-Menten kinetics2,3 seems to be most appropriate for a quantitative comparison of bound with free enzymes. In this way, the data obtained on enzymes adsorbed on colloidal particles can be compared with other methods of immobilization as, for example, by a sol-gel process,30 in macroscopic gels,31 or in polyelectrolyte/protein coacervates.32 Often the value of the Michaelis constant KM is increased considerably if enzymes are immobilized on * To whom correspondence should be addressed. [email protected]. Fax: +49 921 55 2780.

E-mail:

Figure 1. Schematic representation of the spherical polyelectrolyte brushes used in this study. The particles consist of a solid poly(styrene) core onto which long polyelectrolyte chains are grafted. Two systems have been used in this study: (i) particles bearing chains of the weak polyelectrolyte poly(acrylic acid) (annealed brush) and (ii) particles having chains of the strong polyelectrolyte poly(styrene sulfonic acid) (quenched brush). As shown in a previous study, the salt concentration cs inside the brush differs from the salt concentration outside ca markedly if ca is low.34,35 The thickness L of the brush layer depends strongly on ca as has been shown recently.34,35

particles or in macroscopic systems,15,30,31 whereas it remains virtually constant if the immobilization is done by coacervates.32 This points to the onset of diffusional barriers in systems where the enzyme is in direct contact with solid supports. Here we wish to analyze the activity of enzymes immobilized on a new type of colloidal particles. The particles used here are shown schematically in Figure 1. They consist of a solid poly(styrene) core onto which a dense layer of polyelectrolyte chains is grafted.33,34 At low ionic strength, the highly charged brush layer attached to the solid cores is highly stretched by the osmotic pressure of the counterions.34,35 In a recent study, we could demonstrate that proteins like bovine serum albumin (BSA) adsorb onto these particles

10.1021/bm0493584 CCC: $30.25 © 2005 American Chemical Society Published on Web 01/15/2005

Activity of Enzymes in Polyelectrolyte Brushes

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Figure 2. Immobilization of enzymes into spherical polyelectrolyte brushes. The dissolved protein adsorbs spontaneously on the particles if the ionic strength is low. Under these conditions, the adsorption is irreversible and no leaching out of the bound enzymes takes place.36 Enzyme which is left unadsorbed in solution can be removed by ultrafiltration against buffer solution. To probe the enzymatic activity of the immobilized protein, we added an appropriate substrate to the suspension and determined the conversion of the substrate by the enzyme photometrically.

if the ionic strength is low.36 Proteins adsorbed under these conditions (osmotic limit35) will remain within the brush layer even if the suspension is flushed with large amounts of salt solution of the same low ionic strength. If, however, the ionic strength as adjusted through the concentration ca of added salt is raised, BSA is liberated again.36 Figure 2 displays the adsorption experiments in a schematic fashion. A recent study of the BSA thus desorbed from the particles demonstrated that the secondary structure of this protein is not disturbed.37 This result was corroborated by a direct investigation of the secondary structure of several adsorbed proteins through IR spectroscopy.38 It is therefore obvious that denaturation or major conformational changes cannot be the driving force for the strong adsorption. The main driving force was found in the interaction of the negative polyelectrolyte chains with positive patches on the surface of the protein molecule. In this way, the adsorbed protein becomes a counterion of the polyelectrolyte chains.36 A concomitant number of monovalent ions like Na+ is liberated thus increasing the entropy of the system (“counterion release force”; cf. the discussion of this point in ref 36). Pursuant to earlier work,39 we immobilized two enzymes, (i) glucoamylase and (ii) β-glucosidase, on SPB using the method devised previously.36 Various amounts of these enzymes are immobilized on these particles, and the catalytic activity is tested subsequently. Two types of SPB depicted in Figure 1 have been used: (i) particles bearing chains of poly(acrylic acid) (PAA) on their surface and (ii) particles bearing chains of poly(styrene sulfonic acid) (PSS). The grafting density of the surface layer is high so that the linear dimensions of the grafted chains are much higher than their distance on the surface of the particles. The layer consisting of PAA chains hence is an annealed brush whereas PSS chains form a quenched brush.34,35 Both types of particles have been the subject of a number of recent studies.34,35,40-42 In particular, the dependence of the thickness L (see Figure

1) on the concentration ca seems to be well-understood by now.34,35 As a substrate for glucoamylase, 2-chloro-4nitrophenyl-β-D-maltotrioside (CNP-G3) has been used.43 Glucoamylase cleaves the bond between the trioside and the 2-chloro-4-nitrophenol which subsequently can be monitored by UV/vis spectroscopy. 2-Nitrophenyl-β-D-glucopyranoside (NGP) was used as a substrate for β-glucosidase which cleaves the β-D-glucosidic linkage and releases 2-nitrophenol.44 The data obtained on the bound enzymes can subsequently be compared to results obtained from the free enzymes. The choice of these enzymes derives from their technical importance for the cleavage of starch. Starch is degraded by glucoamylase to glucose, and the mechanism of enzymatic activity has been thoroughly studied.45-48 The enzyme belongs to the enzyme class of glucosidases. Moreover, the immobilization of this enzyme on latex particles has been studied by Oh and Kim.15 These authors determined the Michaelis-Menten parameters KM and Vmax for glucoamylase adsorbed on poly(styrene) spheres that had been prepared through co-emulsionpolymerization of styrene and a small amount of styrene sulfonate.49 It is hence interesting to compare the results obtained on these conventional latex particles with data obtained on the spherical polyelectrolyte brushes under consideration here. As a second enzyme, we chose β-glucosidase. The hydrolysis of β-D-glucosidic linkages of glucosides are catalyzed by this enzyme. Hence, both enzymes are of undisputed technical importance, and their immobilization certainly merits further investigations. Experimental Section Materials. Glucoamylase Aspergillus niger (1,4-R-Dglucanglucohydrolase; Fluka) was purified by ultrafiltration against deionized water and freeze-dried. β-Glucosidase from almonds (Sigma) was used without further purification. All other chemicals used here were of analytical grade.

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Spherical Polyelectrolyte Brushes. The synthesis of the spherical polyelectrolyte brushes was done as outlined in refs 33 and 34 by photoemulsionpolymerization. Two types of systems have been synthesized: latex A-SPB bearing chains of poly(acrylic acid) and latex Q-SPB bearing chains of poly(styrene sulfonic acid) (see Figure 1). The core radius of A-SPB and Q-SPB was 58 nm. Both systems were purified through extensive ultrafiltration against pure water. The characterization of the particles was done as described in refs 33-35, and the height L of the brush layer was determined by dynamic light scattering.34,35 For the experiments using glucoamylase, the pH was adjusted to 6.1 by 10 mM N-morpholinoethanesulfonic acid (MES) as described previously.36 In the case of β-glucosidase, the pH was adjusted to 7.1 by 10 mM 3-morpholinopropane sulfonic acid (MOPS). To avoid microbial growth, 2 mM of NaN3 were added to all solutions. The thickness of the brush layer on the particles in these solutions was determined by dynamic light scattering. From these data, a thickness L of the brush layer of the particles of 25 nm was deduced for the annealed SPB A-SPB. For the quenched system, we obtained 93 nm. Adsorption Experiments. Given amounts of glucoamylase (Fluka) and β-glucosidase (Sigma) were dissolved respectively in aqueous 10 mM MES and 10mM MOPS solutions, added to the solutions of the SPBs, and stirred for 24 h at low temperature to avoid microbial growth. The surplus of nonadsorbed enzyme is then removed by ultrafiltration as described recently.36 The amount of nonadsorbed enzyme was determined spectroscopically from the eluate which in turn gives the amount of bound enzyme (extinction coefficients at 278 nm 171 800 M-1 cm-1 for glucoamylase and 76 200 M-1 cm-1 for β-glucosidase). The amount of bound enzyme increases if its concentration in the solution is raised.36 Enzyme Activity. The activity of the free and the bound enzymes was monitored by the assay shown in Figure 3. After immobilization of the enzyme on the particles, the suspensions were diluted to 0.01 g/L, and solutions of the appropriate substrate were added. The concentration of CNPG3 was varied between 0.63 and 5 mM. For β-glucosidase, the concentration of NGP was varied between 1.3 and 10.5 mM because of the lower extinction coefficient of the chromophore. The absorption was measured by UV spectroscopy at 405 nm for typically 10 min. The rate of cleavage V could be determined from the increase of extinction with time. After a short induction period, the increase ∆E/∆t became virtually constant. Figure 4 shows a plot of ∆E/∆t as a function of time. It can be seen that the period of a steady state extends over 8-9 min. Data were taken for further evaluation only if ∆E/∆t remained constant within the given limits of error for an extended period of time. The average of ∆E/∆t taken over this time was converted to the rate V of enzymatic cleavage using the extinction coefficient of 2-chloro-4-nitrophenol (14.9 L/(mmol cm)) or 2-nitrophenol (3.5 L/(mmol cm)) determined photometrically at 405 nm. Control experiments indicated that the absorption of the substrate at 405 nm can be neglected. To monitor the activity of the free enzyme, the appropriate substrate was added to solutions of enzyme (10-3 g/L) in

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Figure 3. Scheme of the reactions used for testing the activity of the enzymes. (a) The substrate CNP-G3 is cleaved in water by the enzyme glucoamylase. The concentration of the resulting 2-chloro4-nitrophenol is monitored photometrically (see Figure 4). (b) The substrate NGP is cleaved in water by the enzyme β-glucosidase. The concentration of the resulting 2-nitrophenol is monitored photometrically (see Figure 4).

the respective buffer solution. The rate of cleavage was measured and evaluated in the same way. Results and Discussion The analysis presented here consists of two steps: First the adsorption of the two enzymes onto the SPB particles is investigated and the amount of enzyme bound in this process is determined. Then the enzymatic activity of the charged particles is measured and compared to results obtained on other supports. Adsorption of Enzymes. The process of adsorption is monitored as shown schematically in Figure 2: Aqueous solutions of the SPB with low ionic strength (7 mM) are


Figure 4. Monitoring the cleavage of the substrate by β-glucosidase immobilized on annealed brush particles. Parameter of the different curves is the concentration [S] of substrate. The change of extinction with time is plotted against time. (top) free β-glucosidase. Filled squares, 1.3 mM; filled circles, 2.2 mM; triangles, 3.2 mM; squares, 4.3 mM; open circles, 10.5 mM. (bottom) 268 mg of β-glucosidase immobilized per g of annealed brush. Filled squares, 2.1 mM; filled circles, 4.2 mM; triangles, 6.3 mM; squares, 8.5 mM; open circles, 10.5 mM. The plot indicates that the rate of cleavage is constant over 8 to 9 min within the given limit of error; hence, the data could be evaluated in the terms of Michaelis-Menten kinetics. The same conclusion was also found to be valid in case of glucoamylase.

mixed with protein solutions at pH ) 6.1 adjusted by the MES buffer36or pH ) 7.1 with MOPS buffer. These solutions are stirred for 24 h, and the enzyme not bound by the particles is removed by ultrafiltration against a buffer solution of the same ionic strength. In this way, the amount τads of enzyme bound per gram of particles can be determined as the function of the concentration csol of enzyme that is left in the suspension. Figure 5 shows the respective plots obtained for the immobilization of glucoamylase onto the annealed system A-SPB (Figure 5a) and the quenched system Q-SPB (Figure 5b). Figure 6 displays the τads of β-glucosidase on the system A-SPB. A recent analysis of the process of adsorption by smallangle X-ray scattering showed that the protein molecules enter deeply inside the brush and the overall volume of it is increased.42 It should be kept in mind that the adsorption thus induced cannot be described in terms of an equilibrium adsorption isotherm.36 If there would be a true equilibrium of the bound and the free protein under these conditions, the ultrafiltration used for the removal of nonadsorbed protein would lead to total desorption. This is not the case, and Figures 5 and 6 demonstrate that a certain fraction of the enzyme is firmly bound to the particles during the process of adsorption. The modeling of the process of adsorption must therefore be divided into two distinct steps: (a) the first step is an equilibration of the free and the bound protein

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Figure 5. Adsorption of glucoamylase into spherical polyelectrolyte brushes. (a) Annealed brush particles bearing chains of poly(acrylic acid). (b) Quenched brush particles bearing chains of poly(styrene sulfonic acid). The solid lines show the fit of the respective data by eq 1. The respective data characterizing the strength of interaction are gathered in Table 1.

Figure 6. Adsorption of β-glucosidase on annealed brush particles. The solid lines show the fit of the experimental data. The respective data characterizing the strength of interaction are gathered in Table 1. The arrows mark the amount of adsorbed enzyme used for the kinetic investigation.

and (b) the adsorbed protein is rearranged within the brush so that the process of desorption becomes slow on the time scale of the present experiment.36 The first step must be related to a conventional adsorption equilibrium. The previous analysis of the adsorption of BSA onto SPB suggested that a model based on the usual BET isotherm gives the best description of data.36 The model also described the adsorption of glucoamylase to either the annealed or the quenched brush (Figure 6). However, in case of the enzyme β-glucosidase with a globular structure,50 the model had to be adapted. Here the data could be described by the conventional Langmuir-Freundlich adsorption iso-

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Table 1. Modeling of the Adsorption of BSA to Spherical Polyelectrolyte Brushes According to Eq 1a τads,M wads SPB enzyme [mg/g SPB] z [mL/mg] A-SPB glucoamylase A-SPB β-glucosidase Q-SPB glucoamylase

70 ( 10 800 ( 100 70 ( 20

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n

6 ( 2 0.19 ( 0.02

1 0.44 ( 0.06 6 ( 3 0.10 ( 0.01 1

a A-SPB, annealed spherical polyelectrolyte brush bearing poly(acrylic acid); Q-SPB, quenched spherical polyelectrolyte brush bearing poly(styrene sulfonic acid); τads,M, maximum mass of protein molecules per unit mass of particles for the first adsorbed layer; z and wads, parameters characterizing strength of adsorption in eq 1.

therm. Compared to the Langmuir isotherm csol has to be transformed to c1/n sol This transformation takes into account that the adsorption energy could depend on the adsorption degree. Hence, the model described in ref 36 was subjected the same transformation. With this expression, all data could be modeled. The resulting expression reads51 zwadsc1/n τads sol ) τads,M (1 - w c1/n)[1 + (z - 1)w c1/n] ads sol ads sol

(1)

where τads,M denotes the maximum mass of protein needed for the strongest adsorbed layer and z wadscsol with z > 1 is the probability of the adsorption of a protein molecule onto this layer. The probability for the adsorption of subsequent layers is only wadscsol. Hence, τads as function of csol may be described in terms of four adjustable parameters τads,M, wads, z, and n.36,51 The previous experiments did show that in the case of the conformational adaptive protein BSA52 the parameter n can be set to 1.36 In other words, the adsorption energy seems to be unaffected by the adsorption degree if the protein possesses a rather flexible tertiary structure. Glucoamylase is a dumbbell-shaped molecule containing a starch-binding domain that is connected via a linker to the catalytic domain.53,54 Here the special architecture of the enzyme seem to also allow some flexibility during the adsorption process as the experimental data in Figure 5 could be well-described under the assumption n ) 1. However, the adsorption of globular proteins as β-glucosidase55 with a rather rigid conformation cannot be described under this assumption. Due to the missing flexibility of the conformation, the adsorption energy might depend strongly on the amount of already adsorbed protein molecules. Hence, it seems that the classification of proteins by Norde and coworkers as either “hard” or “soft” with regard to their adsorption behavior may apply here too.11 The solid line in Figure 6 demonstrates that eq 1 gives an excellent description for β-glucosidase with n ) 0.44. Moreover, fitting recent data38 obtained with the globular enzyme ribonuclease A by eq 1 indicated n ) 0.5. The exponent found here seems therefore to be a reasonable value. Table 1 gathers all data obtained by description of the experimental data. Two points are worth mentioning in this context: (i) There is an excellent stability of the charged particles against flocculation or coagulation. As already shown in ref 36, the colloidal stability is not impeded even at a high loading with BSA. The same is observed here when the SPB are loaded with glucoamylase or β-glucosidase. The stability is certainly due to long polyelectrolyte chains still sticking out even when

Figure 7. Schematic representation of the protein molecules embedded into the spherical polyelectrolyte brush. The diagram shows right in scale the dimension of a typical annealed brush system. The distribution of the adsorbed proteins within the brush layer could be stated by SAXS measurements for BSA and RNase A. Taken from ref 42.

considerable amounts of enzyme are adsorbed onto the particle. It is the necessary prerequisite for the subsequent analysis of the enzymatic activity, of course. (ii) The loading of the spherical polyelectrolyte brushes with proteins and enzymes is very high and the biomolecules penetrate deeply into the surface layer. This could be shown by an analysis of the loaded particles by small-angle X-ray scattering.42 Figure 7 displays the main result of this study obtained for the case of adsorbed ribonuclease A. The enzyme is dispersed throughout the brush layer as expected from the high amount of adsorbed protein. It is obvious that the proteins must interact at this rather high density which leads to a swelling of the brush layer.42 This fact must be taken into account when comparing the activity of the bound enzymes to the free enzymes farther below. Enzymatic ActiWity. The enzymatic activity was evaluated in terms of the classical Michaelis-Menten kinetics2,3 V)

Vmax[S] KM + [S]

where KM is the Michaelis constant and Vmax is the maximum rate attained at infinite concentration of substrate. The maximum rate Vmax divided by the concentration [E] of the enzyme gives kcat which provides a measure for the activity of the enzyme.3 Figures 8 and 10 show the respective data obtained for the free enzyme together with the data obtained for the annealed system A-SPB immobilized enzymes. Figure 9 shows the respective data obtained for the quenched system Q-SPB. All sets of data have been rendered in terms of the Lineweaver-Burk plot,3 which shows the salient point of the analysis in a clear fashion. For the evaluation of the data, however, a nonlinear fit procedure has been used. Figures 8 and 10 demonstrate that eq 2 describes the data of the free enzyme very well. For the free glucoamylase, we find KM ) 9 ( 3 mmol/L, whereas kcat ) 7 mmol/(g min). In the case of β-glucosidase, we find KM ) 6 ( 1 mmol/L, whereas kcat ) 10 mmol/(g min). The data obtained for all bound enzyme are well described in terms of the Michaelis-Menten expression eq 2 (see Figure 8). For both the annealed as well as for the quenched system, straight lines are obtained, and the parameters KM and Vmax can be obtained in sufficient


Biomacromolecules, Vol. 6, No. 2, 2005 953 Table 2. Activity of Glucoamylase Bound to Annealed Spherical Polyelectrolyte Brushesa

Figure 8. Lineweaver-Burk plots of the enzymatic activity of the free and the bound glucoamylase bound to the annealed brush particles A-SPB. The filled circles denote the data obtained for the free enzyme (concentration: 0.001 g/L). Squares, 28 mg of enzyme per g of particles; crosses, 80 mg/g; triangles, 204 mg/g; open circles, 232 mg/g. The values of KM and vmax following from this analysis are gathered in Table 2.

cm [mg GA/g SPB]

KM [mmol/L]

vmax [mmol/(L min)]

kcat [mmol/(g min)]

0 (free enzyme) 23 28 80 97 108 160 204 232

9(3 10 ( 3 11 ( 3 13 ( 3 18 ( 3 18 ( 3 15 ( 3 15 ( 3 18 ( 3

0.5 × 10-2 0.2 × 10-2 0.5 × 10-2 1.7 × 10-2 2.6 × 10-2 2.3 × 10-2 4.7 × 10-2 3.1 × 10-2 4.8 × 10-2

7 0.6 1.1 1.1 1.0 1.5 1.2 0.7 0.9

a c , amount of immobilized enzyme per unit mass of particles; K and m M vmax, see eq 2.

Table 3. Activity of Glucoamylase Bound to Quenched Spherical Polyelectrolyte Brushesa

cm [mg GA/g SPB]

KM [mmol/L]

vmax [mmol/(L min)]

kcat [mmol/(g min)]

0 (free enzyme) 15 47 48 79 85 96

9(3 11 ( 3 14 ( 3 14 ( 3 16 ( 3 16 ( 3 13 ( 3

0.5 × 10-2 0.5 × 10-2 1.0 × 10-2 1.1 × 10-2 1.8 × 10-2 2.2 × 10-2 1.4 × 10-2

7 1.7 1.0 1.1 1.2 1.5 0.9


Figure 9. Lineweaver-Burk plots of the enzymatic activity of the enzyme glucoamylase bound to the quenched brush particles Q-SPB. The amount of bound enzyme per mass of particle was as follows: circles, 15 mg/g; triangles, 47 mg/g; squares, 79 mg/g. The values of KM and vmax following from this analysis are gathered in Table 3.

Figure 10. Lineweaver-Burk plots of the enzymatic activity of the enzyme β-glucosidase bound to the annealed brush particles A-SPB. The filled circles denote the data obtained for the free enzyme (concentration: 0.001 g/L). Squares, 32 mg of enzyme per g of particles; triangles, 268 mg/g; open circles, 580 mg/g. The values of KM and vmax following from this analysis are gathered in Table 4.

accuracy. This is a nontrivial finding since the concentration of the enzyme molecules within the brush layer is much higher as the enzyme concentration used. The respective data are gathered in Tables 2-4. The results demonstrate that the enzyme must remain in its native active state even if adsorbed onto the SPB. This is

in accord with our previous findings showing that the secondary structure of the desorbed proteins is practically unchanged.37 Additional experiments demonstrated that the activity remains unchanged over a considerable period of time. Moreover, no leaching out of the enzyme took place during the reaction. This is a central point of the present analysis since it demonstrates that only bound enzymes catalyze the reaction, not free enzymes that may have desorbed from the particles. Hence, the SPB used here can be regarded as “nanoreactors”: they allow to immobilize the enzymes and provide a stable support. On the other hand, the high surface area of the nanoparticles are devoid of any diffusion barrier that may hamper the enzymatic activity in macroscopic supports. In the following, we shall discuss the kinetic parameters obtained for both enzymes. Glucoamylase. Figure 8 shows the analysis for the data obtained for glucoamylase adsorbed on the annealed brush A-SPB. It demonstrates that the intercept with the abscissa is nearly same for all sets of data. KM increases slightly for the bound enzyme as compared to the free enzyme if large amounts are immobilized. Table 2 gathers the respective data. The same conclusion can be drawn for this enzyme adsorbed on the quenched brush SPB Q-SPB (see Figure 9 and Table 3). This demonstrates that the binding constant embodied in KM is not altered strongly by adsorption unless high amounts of the enzyme are bound to the particles. In that case, the mutual interaction between the immobilized proteins within the brush layer may become important. Moreover, the basic prerequisites of the Michaelis-Menten kinetics may not be fully given anymore. For high amounts of bound glucoamylase, the concentration of substrate molecules may not exceed

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Table 4. Activity of β-Glucosidase Bound to Annealed Spherical Polyelectrolyte Brushesa

cm [mg Glu/g SPB]

KM [mmol/L]

vmax [mmol/(L min)]

kcat [mmol/(g min)]

0 (free enzyme) 32 53 219 268 286 312 324 447 452 580

6(1 11 ( 1 12 ( 1 7(1 6(1 7(1 6(1 6(1 5(1 5(1 5(1

2.7 × 10-2 1.2 × 10-2 0.6 × 10-2 1.8 × 10-2 2.3 × 10-2 1.7 × 10-2 3.6 × 10-2 4.3 × 10-2 1.8 × 10-2 1.1 × 10-2 3.3 × 10-2

10 4.3 1.4 1.9 4.0 6.1 5.6 7.9 3.0 2.4 4.4


greatly the concentration of enzyme molecules anymore. It should be noted, however, that the increase of KM observed in this case is moderate (see below). Only the constant kcat is smaller (ca. 1 mmol/(g min)) than the value for the free enzyme (7 mmol/(g min)). The lowering of enzyme activity may be brought about by possible diffusion barriers created by the rather dense packing of the glucoamylase within the layer. Other reasons that may be invoked include a slightly different pH within the brush layer brought about by the Donnan effect in these systems.35 It should be kept in mind, however, that kcat has a large error since the errors of KM and Vmax add up when calculating this figure. For the discussion of the activity of bound enzymes it therefore seems to possess less significance than KM. β-Glucosidase. As can be seen in Figure 10 and Table 4, the KM values decrease slightly with increasing amount of adsorbed enzyme. This is in opposite to the observation made for glucoamylase. The change of KM, however, is still rather small. The turnover constant kcat is approximately the same but this data is afflicted by a large error as already stated above. Hence, adsorbed β-glucosidase seems to keep its catalytic activity practically at the same level as observed for the free enzyme. All data obtained so far demonstrate that most of the activity of the bound enzymes is preserved in the adsorbed state. Thus, the Michaelis-constant KM is not changed in a drastic manner. However, the turnover constant kcat is decreased in case of the glucoamylase. In view of the fact that the brush layer on the surface of the core particles is densely packed with protein molecules, a change of the kinetic parameters does not appear unreasonable. The local concentration of enzymes is much higher than in free solution and enzyme/enzyme interaction may impede the enzymatic activity. Hence, we believe that these changes are rather small. The present data indicate clearly that no aggregation of the enzymes or other strong protein/protein interactions can taken place within the brush layer. This would immediately lead to a strong decrease of enzymatic activity, of course. Hence, the present data together with previous studies of the secondary38 and tertiary structure37 demonstrate that the spatial structure of the enzymes must be largely preserved.

Our data can directly be compared to a thorough study by Oh and Kim, who analyzed KM of glucoamylase adsorbed onto conventional latex particles.15 Oh and Kim found a sharp rise of KM with an increasing amount of immobilized enzyme (cf. Table 2 of ref 15 and the discussion of these data). These authors discussed several reasons such as partial damage of the protein structure that may be responsible for the lowering of enzyme activity thus effected. Moreover, the latex particles tend to aggregate if more and more enzyme is bound to their surface. A similar increase was found for a related system55 and points to problems regarding the diffusion of the substrate and the products. Diffusional resistance may also be responsible for the increase observed for enzymes immobilized by a sol-gel process30 or in a cross-linked poly(acrylamide) gel.31 The results reported here demonstrate, however, that the spherical polyelectrolyte brushes can immobilize enzymes as glucoamylase and β-glucosidase without impeding its activity. The clear disadvantage of the present way of immobilization is the necessity of keeping the ionic strength constant. For a well-controlled technical process, however, this problem should be overcome. We therefore believe that the novel particles introduced here merit further test with regard to possible technical applications. Conclusion This study has demonstrated that spherical polyelectrolyte brushes allow glucoamylase as well as β-glucosidase to immobilize with nearly full preservation of their activity. This can be shown for the annealed SPB as well as for the quenched SPB. The activity of the bound enzyme furthermore points to the fact that the native structure of the adsorbed protein is fully preserved in accord with previous findings.37,38 It should be kept in mind that both enzymes under scrutiny here differ with regard to their tertiary structure and shape.50,51,53 Moreover, the high amount of adsorbed protein leads to a rather dense packing within the brush layer (cf. Figure 7). Yet both enzymes retain their activity to a large extent. All findings suggest that the novel principle of enzyme immobilization is of general use. As mentioned above, it is based on the “counterion release force” treated in a quantitative manner recently by von Gru¨nberg and co-workers.56 Further studies will be directed to possible technical applications of these “nanoreactors”. Acknowledgment. Financial support by the Roche Diagnostics Company, the Bundesministerium fu¨r Forschung und Technologie, and by the Deutsche Forschungsgemeinschaft is gratefully acknowledged. References and Notes (1) Physical Chemistry of Biological Interface; Baskin, A., Norde, W., Eds.; Marcel Dekker: New York, 1999. (2) Hartmeier, W. Immobilisierte BiokatalysesEine Einfu¨hrung; SpringerVerlag: New York, 1986. (3) Copeland, R. A. Enyzmes, A Practical Introduction to Structure, Mechanism, and Data Analysis; Wiley-VCH: New York, 2000. (4) Kawaguchi, H. Prog. Polym. Sci. 2000, 25, 1171. (5) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1978, 66, 257. (6) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1978, 66, 266. (7) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1978, 66, 277.

Activity of Enzymes in Polyelectrolyte Brushes (8) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1978, 66, 285. (9) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1978, 66, 295. (10) Peula, J. M.; Callejas, J.; de las Nieves, F. J. In Surface Properties of Biomaterials; West, R., Batts, G., Eds.; Butterworth-Heinemann: Manchester, U.K., 1994. (11) Arai, T.; Norde, W. Colloids Surf. 1990, 51, 1. (12) Yoon, J.-Y.; Kim, J.-H.; Kim, W.-S. J. Colloid Interface Sci. 1996, 177, 613. (13) Norde, W.; Giacomelli, C. E. Macromol. Symp. 1999, 145, 125. (14) Giacomelli, C. E.; Vermeer, A. W. P.; Norde, W. J. Colloid Interface Sci. 2000, 231, 283. (15) Oh, J.-T.; Kim, J.-H. Enzyme Microb. Technol. 2000, 27, 356. (16) Chakarova, S. D.; Carlson, A. D. Phys. ReV. E 2004, 69, 021907. (17) Wertz, C. F.; Santore, M. M. Langmuir 2001, 17, 3006. (18) Wertz, C. F.; Santore, M. M. Langmuir 2002, 18, 706. (19) Wertz, C. F.; Santore, M. M. Langmuir 2002, 18, 1190. (20) Czeslik, C.; Winter, R. Phys. Chem. Chem. Phys. 2001, 3, 235. (21) Jackler, G.; Steitz, R.; Czeslik, C. Langmuir 2002, 18, 6565. (22) Czeslik, C.; Royer, C.; Hazlett, T.; Mantulin, W. Biophys. J. 2003, 84, 2533. (23) Fragneto, G.; Su, T. J.; Lu, J. R.; Thomas, R. K.; Rennie, A. R. Phys. Chem. Chem. Phys. 2000, 2, 5214. (24) Caruso, F.; Trau, D.; Mo¨hwald, D.; Renneberg, R. Langmuir 2000, 16, 1485. (25) Caruso, F.; Schu¨ler, C. Langmuir 2000, 16, 9595. (26) Caruso, F.; Schu¨ler, C. Macromol. Rapid Commun. 2000, 21, 750. (27) Caruso, F.; Fiedler, H.; Haage, K. Colloids Surf., A 2000, 169, 287. (28) Lvov, Y.; Caruso, F. Anal. Chem. 2001, 73, 4212. (29) Jin, W.; Shi, X.; Caruso, F. J. Am. Chem. Soc. 2001, 123, 8121. (30) Bhatia, R. B.; Brinker, C. J.; Gupta, A. K.; Singh, A. K. Chem. Mater. 2000, 12, 2434. (31) Sun, S.; Li, X.; Nu, S.; You, X. J. Agric. Food Chem. 1999, 47, 819. (32) Xia, J.; Mattison, K.; Romano, V.; Dubin, P. L.; Muhoberac, B. B. Biopolymers 1996, 41, 359. (33) Guo, X.; Weiss, A.; Ballauff, M. Macromolecules 1999, 32, 6043. (34) Guo, X.; Ballauff, M. Langmuir 2000, 16, 8719. (35) Guo, X.; Ballauff, M. Phys. ReV. E 2001, 64, 051406.

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BM0493584

Activity of Enzymes Immobilized in Colloidal Spherical Polyelectrolyte

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