- Email: [email protected]
Immobilization of trypsin on porous glycidyl methacrylate beads: effects of polymer hydrophilization
Colloids and Surfaces B: Biointerfaces 18 (2000) 277 – 284 www.elsevier.nl/locate/colsurfb
Immobilization of trypsin on porous glycidyl methacrylate beads: effects of polymer hydrophilization Martin Malmsten a,*, Anders Larsson b a
Institute for Surface Chemistry, PO Box 5607, SE-114 86 Stockholm, Sweden b Amersham Pharmacia Biotech AB, SE-751 84 Uppsala, Sweden
Abstract The immobilization of trypsin at porous glycidyl methacrylate (GMA-GDMA) beads was investigated. In particular, the effects of surface modification of the beads through hydrophilic polymers on the amount protein immobilized and on the extent of retained activity after immobilization were adressed. Furthermore, immobilization at unmodified and hydrophilized beads from aqueous solution was compared to that from a water-in-oil microemulsion. It was found that the amount trypsin immobilized at the unmodified GMA-GDMA beads was significantly higher than that at hydrophilized GMA-GDMA beads. However, also the extent of specific activity loss after immobilization was larger for the unmodified than for the hydrophilized beads. Despite the latter, however, the total activity displayed by the hydrophilized beads was comparable to the unmodified beads at best. On the other hand, by peforming the immobilization from the microemulsion a high immobilization yield can be reached even for the hydrophilized beads, which also results in a higher degree of retained activity in the latter case than obtained for immobilization at the unmodified beads. Using this approach therefore resulted in the highest total activity of the trypsin-activated GMA-GDMA beads. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Activity; GMA-GDMA; Hydrophilization; Immobilization; Microemulsion; Trypsin
1. Introduction The successful use of protein immobilization in application areas such as solid phase diagnostics, biosensors, extracorporeal therapy, drug delivery, and biotechnical separation methods requires numerous aspects to be reached. An evident require-
* Corresponding author. Tel.: +46-8-7909910; fax: +46-8208998. E-mail address: [email protected] (M. Malmsten).
ment is a sufficiently strong anchoring of the protein at the substrate, frequently achieved through covalent coupling, in order to eliminate or significantly reduce protein leakage, e.g. through desorption on rinsing or exchange through competitive adsorption on exposure to complex biological fluids. Apart from this, however, there are also other requirements relating to capacity and signal-to-noise ratio. Most importantly, the immobilized molecules should display biological activity also after immobilization. Furthermore, in order to reduce problems with background signal, false positive answers, etc.,
0927-7765/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 9 9 ) 0 0 1 5 3 - 8
278
M. Malmsten, A. Larsson / Colloids and Surfaces B: Biointerfaces 18 (2000) 277–284
unspecific binding of, e.g. the substrate in an enzyme assay or process unit, or antigens in an immunoassay, should be kept at a minimum. Many proteins undergo conformational changes on adsorption. The extent of this process depends on a number of factors, including the protein–surface interaction, interfacial crowding, and the protein structural stability, the latter modulated, e.g. by pH and electrolyte concentration [1–3]. Although some proteins, e.g. certain lipases, may be activated by such interfacial conformational changes, many proteins tend to loose their biological activity to at least some extent from this process. On covalent attachment of a protein to a substrate, the situation is somewhat more complex than that on physical adsorption. Nevertheless, the non-covalent adsorption/binding still constitutes the first step also in a covalent immobilization process, and therefore adsorptioninduced conformational changes and resulting activity loss is likely to be similar, or at least related. For a given protein system to be used immobilized in an assay or a bioreactor, most parameters are given, and can not be widely adjusted. On the other hand, the protein – surface interaction can be varied, e.g. through the choice of substrate materials or through surface modification. Of particular interest for numerous biomedical applications is the use of hydrophilic polymers for surface modification [1,4 – 10]. Surface modifications of this type display a low unspecific protein adsorption, which is advantageous, e.g. for increasing the circulation time of intravenously administered colloidal drug carriers, for reducing the occurence of false positive answers in solid phase diagnostics, and for improving the selectivity and capacity in extracorporeal therapy. [1,4]. Due to the presence of an osmotic repulsive interaction opposing protein adsorption to such a polymer-modified surface, and weak attractive interactions of van der Waals, electrostatic and hydrophobic nature, the protein – ‘surface’ interaction is typically less attractive than that between the protein and the underlying surface [1]. Apart from resulting in a reduced driving force for non-covalent adsorption/binding, one could expect immobilization to such coatings to be characterized by a smaller degree of interfacial
conformational changes, and hence in an increased retained specific activity after immobilization. Indeed, such increased retained activity has been found previously (e.g. 7, 8). Since the steric repulsion between the protein and the hydrophilic polymer layer depends on the solvency, the protein–‘surface’ interaction can be controlled through choice of solvent [9,10]. Thus, if the protein immobilization is performed at poor solvency conditions for the polymer, the steric repulsion is reduced or eliminated, and therefore, a higher reaction yield can be obtained. Such poor solvency conditions can be obtained, e.g. by changing the temperature, by cosolute additions, or by performing the immobilization from an organic solvent, e.g. a water-in-oil microemulsion [9–11]. After immobilization, the solvent can be changed into the (aqueous) solution to be used in the application, which typically corresponds to fairly good solvency conditions for the hydrophilizing polymer coating. At this stage, the coating is once more protein rejecting. With this approach, it should be possible to combine a reasonably high immobilization yield with a retained specific activity that is better than that reached by immobilization directly at the underlying substrate material. In the present investigation, we have studied the immobilization of trypsin at porous cross-linked glycidyl methacrylate (GMA-GDMA) beads, both since trypsin immobilization in itself is of considerable academic interest [12–40] and since such systems could be expected to be of practical interest, particularly in bioprocessing [13,37,39]. More specifically, immobilization at unmodified beads and beads hydrophilized by coupling of water-soluble polymers to the glycidyl epoxides was compared, as was the immobilization at these beads from aqeous solution and from a water-inoil microemulsion.
2. Experimental
2.1. Materials Laboratory preparations of porous poly(glycodyl methacrylate-co-1,3-dimethacrylate) GMA-
M. Malmsten, A. Larsson / Colloids and Surfaces B: Biointerfaces 18 (2000) 277–284
GDMA beads, 125 – 315 mm sieve fraction, were obtained from Amersham Pharmacia Biotech AB, Sweden. These beads have a mean pore diameter of 58 nm (from mercury porosimetry), and a specific surface area of 47 m2/g (BET/N2), except for the mPEO-NH2 surface modification and the epoxide group hydrolysis experiments, in which the beads used had a mean pore diameter of 460 nm, and a specific surface area of 10 m2/g. The amount of epoxide groups of the beads was determined by reaction with sodium thiosulfate followed by titration of the hydroxide ions formed. For surface modification, a number of hydrophilic polymers were used, i.e. dextran T40 (Mw : 40 kDa), dextran AB (Mw :500 kDa), phenyldextran (laboratory preparation; Mw :40 kDa) (all from Amersham Pharmacia Biotech AB, Sweden), hydroxyethylcellulose (HEC; Mw :90 kDa, Hercules, USA), poly(vinyl alcohol/acetate) (PVA), either 80% hydrolyzed (Mw :10 kDa, 36.062–7, Aldrich, Germany) or 88% hydrolyzed (Mowiol 40–88; Mw : 127 kDa, Hoechst, Germany), and a graft copolymer of poly(ethylene imine) (PEI; Mw :2000 kDa, BASF, Germany) and diepoxypoly(ethylene oxide) (PEO; Mw :6 kDa, Akzo Nobel Surface Chemistry, Sweden), the latter with PEI as the backbone, prepared as described previously [6]. Monomethoxy mPEO5000-NH2 was obtained from Shearwater, USA, whereas crystalline porcine trypsin 4500 K, batch P447H-02 (5300 USP/mg) was obtained from Novo Nordisk A/S, and was used as received. All other chemicals used were of analytical grade.
2.2. Methods In order to reduce trypsin self-digestion, the trypsin immobilization to the GMA-GDMA beads from aqueous solution was performed by dissolving the required amount enzyme in 0.3 M borate buffer, pH 8.0, whereafter the beads (0.14 mg/ml) were added and the suspension mixed by gentle shaking at 37°C overnight. The beads were then cleaned with the buffer solution, and stored in buffer/ethanol for a maximum period of 1 week prior to activity measurements. For hydrophilization, 15.8 mg dry beads per ml polymer solution were used. The polymer concentrations for the
279
hydrophilization step were chosen to ensure plateau conditions in the respective polymer adsorption isotherm (not shown). After immobilization, the polymer layer was activated by epichlorohydrin [41–43], except for the PEI-PEO copolymer, which already contained epoxy groups at the end of the PEO chains. For the microemulsion procedure, a water-in-oil microemulsion consisting of 8 wt% aqueous phase (140 mg/ml trypsin in 0.3 M borate buffer, pH 8.0), 8 wt% aerosol OT (AOT), and 84 wt% isooctane, was used [6]. Trypsin was dissolved in the aqueous phase to the desired concentration, whereafter a solution of the AOT solubilized in the oil was added. This mixture was vortexed until a clear microemulsion was obtained. Note, however, that the stability of the microemulsion depends on both the electrolyte and protein concentration. For the borate buffer used in the present investigation, a maximum of about 15 mg/ml of trypsin could be incorporated into the microemulsion. At higher protein concentrations, an emulsion was formed, which restricted the protein concentration in the microemulsion immobilization to below this limit. The microemulsion was then added to the polymer coated beads (0.2 g beads per g microemulsion). After immobilization, the microemulsion was removed by rinsing with buffer. The stability of both trypsin and other enzymes in microemulsions was previously investigated and found to be adequate ([44] and references therein). For the mPEO5000-NH2 surface modification, mPEO5000-NH2 was allowed to prereact with GMA-GDMA beads overnight, and after rinsing this was followed by standard trypsin immobilization from aqueous solution (described above) at 100 mg/ml. The activity of immobilized trypsin was monitored by hydrolyzing N-benzoyl-arginine-ethyl ester (BAEE) (0.145 M) in 0.01 M Tris plus 0.02 M CaCl2, pH 8.0, with trypsin at 25°C in a pH-stat (Metrohm 670 Titroprocessor), and measuring the amount NaOH (0.1 M) needed to neutralize the carboxyl groups generated. The amount trypsin immobilized was determined by amino acid analysis after extensive hydrolysis at the Amino Acid Analysis Center, University of Uppsala.
280
M. Malmsten, A. Larsson / Colloids and Surfaces B: Biointerfaces 18 (2000) 277–284
3. Results and discussion
3.1. Immobilization from aqueous solution at unmodified beads Due to the presence of epoxy groups at the surface of the porous beads, trypsin is easily covalently immobilized at GMA-GDMA beads. Therefore, the amount protein covalently immobilized is generally quite high, even at fairly short immobilization times. As can be inferred from Fig. 1, the immobilization isotherm is of rather high affinity type in the case of unmodified GMAGDMA beads, resulting in quite high amounts immobilized at plateau conditions in the isotherm. Based on the BET surface area the surface concentration of immobilized trypsin at high concentration amounts to about 1.8 mg/m2. This is lower than the saturation adsorption of this protein at a number of hydrophobic surfaces (:2.5 –4.5 mg/
Fig. 1. Effects of the trypsin concentration on its immobilization at unmodified (circles) or dextran-coated (squares) GMAGDMA beads regarding the amount immobilized (a), and the specific activity displayed (b).
m2 [22,24]), but still suggests that the trypsin is indeed fairly crowded at the surface. [It should be noted that there are variations in the amount trypsin immobilized expressed in mass per unit area between different bead batches. This indicates that one should be somewhat cautious in scaling the amount protein immobilized with the BET surface area, since artefacts related to differences in pore volume accessibility restrictions for the protein and the BET probe gas can not be excluded.] Although a very high amount protein immobilized, and also a high total activity, can be reached simply by increasing the protein concentration in the immobilization step, this approach is less attractive from a practical and economic perspective. Instead, it is interesting to maximize the performance at low trypsin concentrations. Although the amount immobilized decreases considerably on decreasing the protein concentration, a considerable amount trypsin can still be immobilized already at a protein concentration of 10 mg/ml. Despite the relatively high amount immobilized also under the latter conditions, however, the total activity displayed by these beads is not very high. The reason for this is that the immobilized trypsin molecules display quite low specific activity. A low specific activity of immobilized trypsin is not unusual for polymeric materials, but has rather been observed in numerous previous investigations [16,17,20–22,24,25,27,30 –34,36]. Note, however, that the extent of activity loss on immobilization varies considerably between different investigations. As an illustration of this, Turkova et al. investigated the immobilization of trypsin at glycidyl methacrylate beads, and found very high-retained activity even at quite low amounts trypsin immobilized [40]. In the present investigation, on the other hand, a low retained activity after immobilization at GMA-GDMA beads was found. The origin of the difference between the present findings and those of this previous investigation is unknown at present, but may be due to differences in the bead and protein preparations, or in the coupling procedures used. The reason for the frequently found low retained activity after immobilization is probably a combined effect of interfacial conformational
M. Malmsten, A. Larsson / Colloids and Surfaces B: Biointerfaces 18 (2000) 277–284
Fig. 2. The amount trypsin immobilized at GMA-GDMA beads as a function of the epoxide group density.
change leading to activity loss [1 – 3], orientation-related active site access restrictions [45,46], mass transport limitations in the dense interfacial protein layer, trypsin self digestion, etc. Of these, effects due to the protein – surface interaction are probably of special importance. Thus, when a protein adsorbs at a surface, there is frequently a change in tertiary and/or secondary structure (e.g. loss of a-helix). Often, although not always, this conformational change results in a reduced specific activity [1–3]. In this particular system, this effect can be expected to be of particular importance, since apart from attractive physicochemical protein–surface interactions of hydrophobic and van der Waals nature, the high epoxide group density offers ample opportunity for covalent multi-point attachment through reactions with amine or thiol-containing amino acid residues. In order to illustrate the importance of the epoxide group density on the immobilization yield, Fig. 2 shows the amount trypsin immobilized at various epoxide group densities, the latter achieved through hydrolysis at different pH values. As can be seen, the amount immobilized decreases with a decreasing epoxide group density, which suggests the importance of covalent attachment as immobilization mechanism. Also after quantitative hydrolysis of the epoxide groups, however, a considerable amount trypsin is bound to the beads, which shows that also physical adsorption is important for trypsin immobilization at these beads. Quantitatively, these findings suggest that up to 50% of the protein may be physically adsorbed rather than
281
covalently immobilized at the conditions discussed further below. Given this, as well as the importance of both physical attractive interactions and excessive covalent immobilization for the specific activity loss of immobilized trypsin, an interesting approach for reaching an increased retained specific activity after immobilization is to modify the surface with a hydrophilic and uncharged polymer. Since the layers formed by such polymers are typically very rich in water [47], the van der Waals interaction between a protein and such a surface coating is small [48]. Furthermore, since the layers are uncharged and hydrophilic, electrostatic and hydrophobic interactions are largely absent. Combined with the steric repulsive interaction between such a surface layer and proteins, this means that surface modification through hydrophilic polymers constitutes a way to reduce the protein–surface attraction. Apart from this, such a layer would have a much lower reactive group density, which may further reduce specific activity loss on immobilization.
3.2. Immobilization from aqueous solution at hydrophilized beads Due to the absence of strong attractive interactions between the surface coating and the protein, and to the steric repulsion acting between the polymer layer and the protein, the immobilization yield for such a system may be a problem. This is illustrated in Fig. 3, which shows the amount trypsin immobilized at the GMA-GDMA surface as a function of the PEO concentration used for the surface hydrophilization. As is clearly shown, the amount protein immobilized decreases strongly with the pretreatment concentration, which is an effect of the PEO chain density on the protein rejection, as discussed previously [47]. This behaviour is displayed also for immobilization at the fully formed hydrophilic layer, as shown in Fig. 1 for dextran. Thus, the amount trypsin immobilized is much lower for dextran-coated than for unmodified beads. Although the amount immobilized is lower for hydrophilized than for unmodified beads, it is possible that the total activity is equally good or
282
M. Malmsten, A. Larsson / Colloids and Surfaces B: Biointerfaces 18 (2000) 277–284
Fig. 3. Effect of the concentration of mPEO5000-NH2 prereacted with the GMA-GDMA beads on the subsequent trypsin immobilization.
even better, due to a higher retained specific activity. If this is the case, the hydrophilized beads are better than the unmodified ones in that the unspecific adsorption to such a system is generally much lower than for unmodified beads [1,4–6]. Considering this, we immobilized trypsin at a number of hydrophilized GMA-GDMA beads. As can be seen in Table 1, the specific activity is indeed higher for all the hydrophilized beads investigated, and for dextran much higher than for the unmodified beads, although the amounts immobilized in all cases is much lower. In the latter case, the total activity displayed by the hydrophilized beads is comparable to that obtained for the unmodified beads, while for the other polymers, the resulting total activity is lower than that for the unmodified beads.
3.3. Immobilization from microemulsion at hydrophilized beads As discussed above, the protein rejecting prop-
erties of uncharged hydrophilic polymer coatings are well known and due to a combination of a steric repulsive interaction between the polymer layer and the protein, and the lack of strong electrostatic, van der Waals and other attractive interactions [1]. However, the steric interaction depends strongly on the solvency of the polymer [1,48–50]. Therefore, while uncharged flexible polymers are efficient protein rejectants at good solvency conditions, their performance deteriorate at poor solvency conditions due to a contraction of the interfacial layer. With this in mind, several different immobilization techniques based on good-poor solvency cycles have been developed [9,10]. One of these is based on a water-in-oil microemulsion [9]. In such a microemulsion, the protein is present in water droplets dispersed in the oil continuous phase, while the polymer layer experiences poor solvency conditions due to contact with the non-aqueous phase. Therefore, immobilization could be expected to be better from such a microemulsion than from aqueous solution, at least provided that the protein activity is not lost by the contact with surface active agents present in the microemulsion. Indeed, it has been shown before that such a procedure results in a higher immobilization yield than immobilization from aqueous solution [9]. Considering this, we investigated the immobilization of trypsin from such a microemulsion to GMA-GDMA beads hydrophilized through various polymer coatings. As can be seen from Table 2, a significant immobilization yield is obtained for all the coatings, and for all coatings a significantly higher total activity was obtained compared to the unmodified beads. The total activity increase compared to immobilization at the hydrophilized beads from
Table 1 Immobilization of trypsin at hydrophilized GMA-GDMA beads from 0.3 M borate buffer, pH 8. Trypsin concentration 11.8 mg/ml. The specific activity of trypsin in aqueous solution was 50 95 BAEE/mg trypsin Coating
Activity (BAEE/g bead)
Amount (mg/g bead)
Specific activity (BAEE/mg trypsin)
Unmodified HEC PVA 88% Dextran T40
145 64 78 119
40.2 8.4 9.7 8.1
3.6 7.7 8.0 14.6
M. Malmsten, A. Larsson / Colloids and Surfaces B: Biointerfaces 18 (2000) 277–284
283
Table 2 Activity of trypsin immobilized at various hydrophilized GMA-GDMA beads by the microemulsion procedure. Trypsin concentration 11.8 mg/ml. The specific activity of trypsin in aqueous solution was 50 9 5 BAEE/mg trypsin Coating
Activity (BAEE/g bead)
Amount (mg/g bead)
Specific activity (BAEE/mg trypsin)
Unmodified Phenyldextran Dextran AB Dextran T40 HEC PVA 80% PVA 88% PEI-PEO
159 174 229 249 245 207 199 224
32.4 24.0 23.7 23.8 26.1 24.3 29.4 29.7
4.9 7.3 9.5 10.5 9.4 8.5 6.8 7.5
aqueous solution is due to an increased immobilization yield, whereas the total activity improvement compared to the unmodified beads is due to an increased specific activity. Although the results in Table 2 display some variation between the different polymer coatings, which are presently poorly understood and require further studies, the main point is that quailitatively similar results are obtained for a rather wide range of hydrophilic polymer coatings, underlining the generality of the approach. In previous investigations, protein immobilization at hydrophilized surfaces from a microemulsion has been utilized in several contexts. For example, Thomas et al., and Malmsten et al. investigated the immobilization of antibodies in relation to solid phase diagnostics, whereas Moachon et al. investigated the usefulnes of such coatings in extracorporeal therapy [1,5,6]. In these and other studies, the approach taken has shown very low levels of unspecific adsorption, high signal-to-noice-ratio, and superior performance in different biomedical applications. Also the solvency approach has been used before to improve the immobilization yield at hydrophilized surfaces, using either temperature [10] or microemulsion [9] to deteriorate the solvency conditions for the hydrophilic polymer coatings. However, to our knowledge this is the first time that the usefulnes of the approach taken also for bioprocessing is illustrated. Examples of such applications where this immobilization procedure holds promise include manufacturing of protein hydrolysates for reducing allergic problems with childrens dairy
products, self-cleaning ultrafiltration units, and wound cleaning compression textiles, just to mention a few.
4. Summary The immobilization of trypsin at porous glycidyl methacrylate (GMA-GDMA) beads was investigated regarding immobilization yield and retained activity after immobilization. In particular, the immobilization at unmodified GMAGDMA beads was compared to that at beads hydrophilized by various polymer coatings, as was the immobilization at these beads from aqueous solution and a water-in-oil microemulsion. While the immobilization yield was lower for the hydrophilized than for the unmodified beads, the retained specific activity was larger. By performing the immobilization at hydrophilized beads from the microemulsion, a higher yield than obtained by immobilization at hydrophilized beads from aqueous solution could be combined with a higher specific activity than obtained by immobilization at the unmodified beads, thereby resulting in improved total activities. At the same time, however, the (water-in-oil) microemulsion stability area may, as in the present case, limit the protein concentration, which can be used in the immobilization procedure, particularly at high electrolyte concentration. This emphasizes the need in future work to identify microemulsions with a larger protein solubilization capacity.
284
M. Malmsten, A. Larsson / Colloids and Surfaces B: Biointerfaces 18 (2000) 277–284
Acknowledgements Mats Gruvega˚rd is thanked for bead preparation and analysis, while Eva Lundgren is acknowledged for performing the experimental bead functionalization work. Furthermore, the authors would like to thank Mats Gruvega˚rd and PerMikael A, berg, Amersham Pharmacia Biotech, for fruitful discussion. This study was part of the Brite Euram project ‘Solid Matrices for Bioprocesses’ (BE95-1075), and was funded by EU and the Swedish National Board for Industrial and Technical Development (NUTEK).
References [1] M. Malmsten (Eds.), Biopolymers at Interfaces, Surfactant Science Series, vol. 75, Marcel Dekker, New York, 1998. [2] M. Malmsten, J. Colloid Interface Sci. 207 (1998) 186. [3] A. Kondo, F. Murakami, M. Kawagoe, K. Higashitani, Appl. Microbiol. Biotechnol. 39 (1993) 726. [4] J.M. Harris, Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications, Plenum Press, New York, 1992. [5] M. Malmsten, B. Lassen, K. Holmberg, V. Thomas, G. Quash, J. Colloid Interface Sci. 177 (1996) 70. [6] V. Thomas, K. Bergstro¨m, G. Quash, K. Holmberg, Colloid Surf. A 77 (1993) 125. [7] J.M. Harris, K. Yoshinaga, J. Bioact. Compat. Polym. 4 (1989) 281. [8] M.B. Stark, K. Holmberg, Biotech. Bioeng. 34 (1989) 942. [9] K. Bergstro¨m, K. Holmberg, Colloids Surf. 63 (1992) 273. [10] F. Tiberg, C. Brink, M. Hellsten, K. Holmberg, Colloid Polym. Sci. 270 (1992) 1188. [11] M. Malmsten, B. Lindman, Langmuir 6 (1990) 357. [12] D. Bharadwaj, A. Mukherjee, M.S. Roy, R.N. Hati, R.N. Mukherjee, Ind. J. Biochem. Biophys. 29 (1992) 375. [13] P.C. Lorenzen, E. Schlimme, Z. Erna¨rungswiss. 34 (1995) 118. [14] R. Batra, R. Tyagia, M.N. Gupta, Biocatal. Biotransform. 15 (1997) 101. [15] M.A. Cohenford, P.F. Santoro, J.C. Urbanowski, J.A. Dain, Biotechnol. Bioeng. 28 (1986) 736. [16] A. Kumar, M.N. Gupta, J. Mol. Catal. B 5 (1998) 289. [17] H.A. Chase, Y. Yang, Biotechnol. Appl. Biochem. 27 (1998) 205. [18] P. Wang, T.G. Hill, C.A. Wartchow, et al., J. Am. Chem. Soc. 114 (1992) 378. [19] E.A. Kulik, K. Kato, M.I. Ivanchenko, Y. Ikada, Biomaterials 14 (1993) 763.
[20] M. Alves Da Silva, M.H. Gil, J. Guiomar, et al., Radiat. Phys. Chem. 36 (1990) 589. [21] S. Devi, J.T. Guthrie, C.G. Beddows, Radiat. Phys. Chem. 36 (1990) 697. [22] D. Lewis, T.L. Whateley, Biomaterials 9 (1988) 71. [23] D. Wu, R.R. Walters, J. Chromatogr. 458 (1988) 169. [24] S. Kamei, M. Okubo, T. Matsumoto, J. Appl. Polym. Sci. 34 (1987) 1439. [25] M.A. Jasim, G.M. Hall, J. Mann, K.D.A. Taylor, J. Chem. Tech. Biotechnol. 40 (1987) 251. [26] J. Bo, X. Ming, Eur. Polym. J. 28 (1992) 827. [27] R.M. Blanco, J.M. Guisan, Enzyme Microb. Technol. 11 (1989) 360. [28] R.M. Blanco, J.J. Calvete, J.M. Guisan, Enzyme Microb. Technol. 11 (1989) 353. [29] H. Dauzenberg, N. Karibyants, S.Y. Zaitsev, Macromol. Rapid Commun. 18 (1997) 175. [30] S. Kamei, M. Okubo, T. Matsuda, T. Matsumoto, Colloid Polym. Sci. 264 (1986) 743. [31] T. Shiroya, N. Tamura, M. Yasui, K. Fujimoto, H. Kawaguchi, Colloids Surf. B 4 (1995) 267. [32] M. Yasui, T. Shiroya, K. Fujimoto, H. Kawaguchi, Colloids Surf. B 8 (1997) 311. [33] E.T. Kang, K.L. Tan, K. Kato, Y. Uyama, Y. Ikada, Macromolecules 29 (1996) 6872. [34] S. Devi, J.T. Guthrie, C.G. Beddows, Radiat. Phys. Chem. 36 (1990) 703. [35] M. Okubo, H. Ahmad, J. Polym. Sci. A 36 (1998) 883. [36] C.G. Beddows, M.H. Gil, J.T. Guthrie, J. Appl. Polym. Sci. 35 (1988) 135. [37] M. Rucka, G. Pozniak, B. Turkiewicz, W. Trochimczuk, Enzyme Microb. Technol. 18 (477) (1996) 477. [38] M. Sardar, R. Agarwal, A. Kumar, M.N. Gupta, Enzyme Microb. Technol. 20 (1997) 361. [39] P.C. Lorenzen, H. Fischer, E. Schlimme, Die Nahrung 38 (1994) 551. [40] J. Turkova, K. Blaha, M. Malanikova, D. Vancurova, F. Svec, J. Kalal, Biochim. Biophys. Acta 524 (1978) 162. [41] J. Porath, N. Fornstedt, J. Chromatogr. 51 (1970) 479. [42] I. Matsumoto, Y. Mizuno, N. Seno, J. Biochem. 85 (1979) 1091. [43] L. Sundberg, J. Porath, J. Chromatogr. 90 (1974) 87. [44] S. Avramiotis, P. Lianos, A. Xenakis, Biocatal. Biotransform. 14 (1997) 299. [45] I.N. Chang, J.N. Herron, Langmuir 11 (1995) 2083. [46] C.S. Lee, G. Belfort, Proc. Natl. Acad. Sci. USA 86 (1989) 8392. [47] M. Malmsten, K. Emoto, J.M. Van Alstine, J. Colloid Interface Sci. 202 (1998) 507. [48] J.N. Israelachvili, Intermolecular Surface Forces, Academic Press, London, 1992. [49] G.J. Fleer, M.A. Cohen Stuart, J.M.H.M. Scheutjens, T. Cosgrove, B. Vincent, Polymers at Interfaces, Chapman and Hall, London, 1993. [50] D.H. Napper, Polymeric Stabilization of Colloidal Dispersions, Academic Press, London, 1983.
Immobilization of trypsin on porous glycidyl methacrylate beads: effects of polymer hydrophilization Martin Malmsten a,*, Anders Larsson b a
Institute for Surface Chemistry, PO Box 5607, SE-114 86 Stockholm, Sweden b Amersham Pharmacia Biotech AB, SE-751 84 Uppsala, Sweden
Abstract The immobilization of trypsin at porous glycidyl methacrylate (GMA-GDMA) beads was investigated. In particular, the effects of surface modification of the beads through hydrophilic polymers on the amount protein immobilized and on the extent of retained activity after immobilization were adressed. Furthermore, immobilization at unmodified and hydrophilized beads from aqueous solution was compared to that from a water-in-oil microemulsion. It was found that the amount trypsin immobilized at the unmodified GMA-GDMA beads was significantly higher than that at hydrophilized GMA-GDMA beads. However, also the extent of specific activity loss after immobilization was larger for the unmodified than for the hydrophilized beads. Despite the latter, however, the total activity displayed by the hydrophilized beads was comparable to the unmodified beads at best. On the other hand, by peforming the immobilization from the microemulsion a high immobilization yield can be reached even for the hydrophilized beads, which also results in a higher degree of retained activity in the latter case than obtained for immobilization at the unmodified beads. Using this approach therefore resulted in the highest total activity of the trypsin-activated GMA-GDMA beads. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Activity; GMA-GDMA; Hydrophilization; Immobilization; Microemulsion; Trypsin
1. Introduction The successful use of protein immobilization in application areas such as solid phase diagnostics, biosensors, extracorporeal therapy, drug delivery, and biotechnical separation methods requires numerous aspects to be reached. An evident require-
* Corresponding author. Tel.: +46-8-7909910; fax: +46-8208998. E-mail address: [email protected] (M. Malmsten).
ment is a sufficiently strong anchoring of the protein at the substrate, frequently achieved through covalent coupling, in order to eliminate or significantly reduce protein leakage, e.g. through desorption on rinsing or exchange through competitive adsorption on exposure to complex biological fluids. Apart from this, however, there are also other requirements relating to capacity and signal-to-noise ratio. Most importantly, the immobilized molecules should display biological activity also after immobilization. Furthermore, in order to reduce problems with background signal, false positive answers, etc.,
0927-7765/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 9 9 ) 0 0 1 5 3 - 8
278
M. Malmsten, A. Larsson / Colloids and Surfaces B: Biointerfaces 18 (2000) 277–284
unspecific binding of, e.g. the substrate in an enzyme assay or process unit, or antigens in an immunoassay, should be kept at a minimum. Many proteins undergo conformational changes on adsorption. The extent of this process depends on a number of factors, including the protein–surface interaction, interfacial crowding, and the protein structural stability, the latter modulated, e.g. by pH and electrolyte concentration [1–3]. Although some proteins, e.g. certain lipases, may be activated by such interfacial conformational changes, many proteins tend to loose their biological activity to at least some extent from this process. On covalent attachment of a protein to a substrate, the situation is somewhat more complex than that on physical adsorption. Nevertheless, the non-covalent adsorption/binding still constitutes the first step also in a covalent immobilization process, and therefore adsorptioninduced conformational changes and resulting activity loss is likely to be similar, or at least related. For a given protein system to be used immobilized in an assay or a bioreactor, most parameters are given, and can not be widely adjusted. On the other hand, the protein – surface interaction can be varied, e.g. through the choice of substrate materials or through surface modification. Of particular interest for numerous biomedical applications is the use of hydrophilic polymers for surface modification [1,4 – 10]. Surface modifications of this type display a low unspecific protein adsorption, which is advantageous, e.g. for increasing the circulation time of intravenously administered colloidal drug carriers, for reducing the occurence of false positive answers in solid phase diagnostics, and for improving the selectivity and capacity in extracorporeal therapy. [1,4]. Due to the presence of an osmotic repulsive interaction opposing protein adsorption to such a polymer-modified surface, and weak attractive interactions of van der Waals, electrostatic and hydrophobic nature, the protein – ‘surface’ interaction is typically less attractive than that between the protein and the underlying surface [1]. Apart from resulting in a reduced driving force for non-covalent adsorption/binding, one could expect immobilization to such coatings to be characterized by a smaller degree of interfacial
conformational changes, and hence in an increased retained specific activity after immobilization. Indeed, such increased retained activity has been found previously (e.g. 7, 8). Since the steric repulsion between the protein and the hydrophilic polymer layer depends on the solvency, the protein–‘surface’ interaction can be controlled through choice of solvent [9,10]. Thus, if the protein immobilization is performed at poor solvency conditions for the polymer, the steric repulsion is reduced or eliminated, and therefore, a higher reaction yield can be obtained. Such poor solvency conditions can be obtained, e.g. by changing the temperature, by cosolute additions, or by performing the immobilization from an organic solvent, e.g. a water-in-oil microemulsion [9–11]. After immobilization, the solvent can be changed into the (aqueous) solution to be used in the application, which typically corresponds to fairly good solvency conditions for the hydrophilizing polymer coating. At this stage, the coating is once more protein rejecting. With this approach, it should be possible to combine a reasonably high immobilization yield with a retained specific activity that is better than that reached by immobilization directly at the underlying substrate material. In the present investigation, we have studied the immobilization of trypsin at porous cross-linked glycidyl methacrylate (GMA-GDMA) beads, both since trypsin immobilization in itself is of considerable academic interest [12–40] and since such systems could be expected to be of practical interest, particularly in bioprocessing [13,37,39]. More specifically, immobilization at unmodified beads and beads hydrophilized by coupling of water-soluble polymers to the glycidyl epoxides was compared, as was the immobilization at these beads from aqeous solution and from a water-inoil microemulsion.
2. Experimental
2.1. Materials Laboratory preparations of porous poly(glycodyl methacrylate-co-1,3-dimethacrylate) GMA-
M. Malmsten, A. Larsson / Colloids and Surfaces B: Biointerfaces 18 (2000) 277–284
GDMA beads, 125 – 315 mm sieve fraction, were obtained from Amersham Pharmacia Biotech AB, Sweden. These beads have a mean pore diameter of 58 nm (from mercury porosimetry), and a specific surface area of 47 m2/g (BET/N2), except for the mPEO-NH2 surface modification and the epoxide group hydrolysis experiments, in which the beads used had a mean pore diameter of 460 nm, and a specific surface area of 10 m2/g. The amount of epoxide groups of the beads was determined by reaction with sodium thiosulfate followed by titration of the hydroxide ions formed. For surface modification, a number of hydrophilic polymers were used, i.e. dextran T40 (Mw : 40 kDa), dextran AB (Mw :500 kDa), phenyldextran (laboratory preparation; Mw :40 kDa) (all from Amersham Pharmacia Biotech AB, Sweden), hydroxyethylcellulose (HEC; Mw :90 kDa, Hercules, USA), poly(vinyl alcohol/acetate) (PVA), either 80% hydrolyzed (Mw :10 kDa, 36.062–7, Aldrich, Germany) or 88% hydrolyzed (Mowiol 40–88; Mw : 127 kDa, Hoechst, Germany), and a graft copolymer of poly(ethylene imine) (PEI; Mw :2000 kDa, BASF, Germany) and diepoxypoly(ethylene oxide) (PEO; Mw :6 kDa, Akzo Nobel Surface Chemistry, Sweden), the latter with PEI as the backbone, prepared as described previously [6]. Monomethoxy mPEO5000-NH2 was obtained from Shearwater, USA, whereas crystalline porcine trypsin 4500 K, batch P447H-02 (5300 USP/mg) was obtained from Novo Nordisk A/S, and was used as received. All other chemicals used were of analytical grade.
2.2. Methods In order to reduce trypsin self-digestion, the trypsin immobilization to the GMA-GDMA beads from aqueous solution was performed by dissolving the required amount enzyme in 0.3 M borate buffer, pH 8.0, whereafter the beads (0.14 mg/ml) were added and the suspension mixed by gentle shaking at 37°C overnight. The beads were then cleaned with the buffer solution, and stored in buffer/ethanol for a maximum period of 1 week prior to activity measurements. For hydrophilization, 15.8 mg dry beads per ml polymer solution were used. The polymer concentrations for the
279
hydrophilization step were chosen to ensure plateau conditions in the respective polymer adsorption isotherm (not shown). After immobilization, the polymer layer was activated by epichlorohydrin [41–43], except for the PEI-PEO copolymer, which already contained epoxy groups at the end of the PEO chains. For the microemulsion procedure, a water-in-oil microemulsion consisting of 8 wt% aqueous phase (140 mg/ml trypsin in 0.3 M borate buffer, pH 8.0), 8 wt% aerosol OT (AOT), and 84 wt% isooctane, was used [6]. Trypsin was dissolved in the aqueous phase to the desired concentration, whereafter a solution of the AOT solubilized in the oil was added. This mixture was vortexed until a clear microemulsion was obtained. Note, however, that the stability of the microemulsion depends on both the electrolyte and protein concentration. For the borate buffer used in the present investigation, a maximum of about 15 mg/ml of trypsin could be incorporated into the microemulsion. At higher protein concentrations, an emulsion was formed, which restricted the protein concentration in the microemulsion immobilization to below this limit. The microemulsion was then added to the polymer coated beads (0.2 g beads per g microemulsion). After immobilization, the microemulsion was removed by rinsing with buffer. The stability of both trypsin and other enzymes in microemulsions was previously investigated and found to be adequate ([44] and references therein). For the mPEO5000-NH2 surface modification, mPEO5000-NH2 was allowed to prereact with GMA-GDMA beads overnight, and after rinsing this was followed by standard trypsin immobilization from aqueous solution (described above) at 100 mg/ml. The activity of immobilized trypsin was monitored by hydrolyzing N-benzoyl-arginine-ethyl ester (BAEE) (0.145 M) in 0.01 M Tris plus 0.02 M CaCl2, pH 8.0, with trypsin at 25°C in a pH-stat (Metrohm 670 Titroprocessor), and measuring the amount NaOH (0.1 M) needed to neutralize the carboxyl groups generated. The amount trypsin immobilized was determined by amino acid analysis after extensive hydrolysis at the Amino Acid Analysis Center, University of Uppsala.
280
M. Malmsten, A. Larsson / Colloids and Surfaces B: Biointerfaces 18 (2000) 277–284
3. Results and discussion
3.1. Immobilization from aqueous solution at unmodified beads Due to the presence of epoxy groups at the surface of the porous beads, trypsin is easily covalently immobilized at GMA-GDMA beads. Therefore, the amount protein covalently immobilized is generally quite high, even at fairly short immobilization times. As can be inferred from Fig. 1, the immobilization isotherm is of rather high affinity type in the case of unmodified GMAGDMA beads, resulting in quite high amounts immobilized at plateau conditions in the isotherm. Based on the BET surface area the surface concentration of immobilized trypsin at high concentration amounts to about 1.8 mg/m2. This is lower than the saturation adsorption of this protein at a number of hydrophobic surfaces (:2.5 –4.5 mg/
Fig. 1. Effects of the trypsin concentration on its immobilization at unmodified (circles) or dextran-coated (squares) GMAGDMA beads regarding the amount immobilized (a), and the specific activity displayed (b).
m2 [22,24]), but still suggests that the trypsin is indeed fairly crowded at the surface. [It should be noted that there are variations in the amount trypsin immobilized expressed in mass per unit area between different bead batches. This indicates that one should be somewhat cautious in scaling the amount protein immobilized with the BET surface area, since artefacts related to differences in pore volume accessibility restrictions for the protein and the BET probe gas can not be excluded.] Although a very high amount protein immobilized, and also a high total activity, can be reached simply by increasing the protein concentration in the immobilization step, this approach is less attractive from a practical and economic perspective. Instead, it is interesting to maximize the performance at low trypsin concentrations. Although the amount immobilized decreases considerably on decreasing the protein concentration, a considerable amount trypsin can still be immobilized already at a protein concentration of 10 mg/ml. Despite the relatively high amount immobilized also under the latter conditions, however, the total activity displayed by these beads is not very high. The reason for this is that the immobilized trypsin molecules display quite low specific activity. A low specific activity of immobilized trypsin is not unusual for polymeric materials, but has rather been observed in numerous previous investigations [16,17,20–22,24,25,27,30 –34,36]. Note, however, that the extent of activity loss on immobilization varies considerably between different investigations. As an illustration of this, Turkova et al. investigated the immobilization of trypsin at glycidyl methacrylate beads, and found very high-retained activity even at quite low amounts trypsin immobilized [40]. In the present investigation, on the other hand, a low retained activity after immobilization at GMA-GDMA beads was found. The origin of the difference between the present findings and those of this previous investigation is unknown at present, but may be due to differences in the bead and protein preparations, or in the coupling procedures used. The reason for the frequently found low retained activity after immobilization is probably a combined effect of interfacial conformational
M. Malmsten, A. Larsson / Colloids and Surfaces B: Biointerfaces 18 (2000) 277–284
Fig. 2. The amount trypsin immobilized at GMA-GDMA beads as a function of the epoxide group density.
change leading to activity loss [1 – 3], orientation-related active site access restrictions [45,46], mass transport limitations in the dense interfacial protein layer, trypsin self digestion, etc. Of these, effects due to the protein – surface interaction are probably of special importance. Thus, when a protein adsorbs at a surface, there is frequently a change in tertiary and/or secondary structure (e.g. loss of a-helix). Often, although not always, this conformational change results in a reduced specific activity [1–3]. In this particular system, this effect can be expected to be of particular importance, since apart from attractive physicochemical protein–surface interactions of hydrophobic and van der Waals nature, the high epoxide group density offers ample opportunity for covalent multi-point attachment through reactions with amine or thiol-containing amino acid residues. In order to illustrate the importance of the epoxide group density on the immobilization yield, Fig. 2 shows the amount trypsin immobilized at various epoxide group densities, the latter achieved through hydrolysis at different pH values. As can be seen, the amount immobilized decreases with a decreasing epoxide group density, which suggests the importance of covalent attachment as immobilization mechanism. Also after quantitative hydrolysis of the epoxide groups, however, a considerable amount trypsin is bound to the beads, which shows that also physical adsorption is important for trypsin immobilization at these beads. Quantitatively, these findings suggest that up to 50% of the protein may be physically adsorbed rather than
281
covalently immobilized at the conditions discussed further below. Given this, as well as the importance of both physical attractive interactions and excessive covalent immobilization for the specific activity loss of immobilized trypsin, an interesting approach for reaching an increased retained specific activity after immobilization is to modify the surface with a hydrophilic and uncharged polymer. Since the layers formed by such polymers are typically very rich in water [47], the van der Waals interaction between a protein and such a surface coating is small [48]. Furthermore, since the layers are uncharged and hydrophilic, electrostatic and hydrophobic interactions are largely absent. Combined with the steric repulsive interaction between such a surface layer and proteins, this means that surface modification through hydrophilic polymers constitutes a way to reduce the protein–surface attraction. Apart from this, such a layer would have a much lower reactive group density, which may further reduce specific activity loss on immobilization.
3.2. Immobilization from aqueous solution at hydrophilized beads Due to the absence of strong attractive interactions between the surface coating and the protein, and to the steric repulsion acting between the polymer layer and the protein, the immobilization yield for such a system may be a problem. This is illustrated in Fig. 3, which shows the amount trypsin immobilized at the GMA-GDMA surface as a function of the PEO concentration used for the surface hydrophilization. As is clearly shown, the amount protein immobilized decreases strongly with the pretreatment concentration, which is an effect of the PEO chain density on the protein rejection, as discussed previously [47]. This behaviour is displayed also for immobilization at the fully formed hydrophilic layer, as shown in Fig. 1 for dextran. Thus, the amount trypsin immobilized is much lower for dextran-coated than for unmodified beads. Although the amount immobilized is lower for hydrophilized than for unmodified beads, it is possible that the total activity is equally good or
282
M. Malmsten, A. Larsson / Colloids and Surfaces B: Biointerfaces 18 (2000) 277–284
Fig. 3. Effect of the concentration of mPEO5000-NH2 prereacted with the GMA-GDMA beads on the subsequent trypsin immobilization.
even better, due to a higher retained specific activity. If this is the case, the hydrophilized beads are better than the unmodified ones in that the unspecific adsorption to such a system is generally much lower than for unmodified beads [1,4–6]. Considering this, we immobilized trypsin at a number of hydrophilized GMA-GDMA beads. As can be seen in Table 1, the specific activity is indeed higher for all the hydrophilized beads investigated, and for dextran much higher than for the unmodified beads, although the amounts immobilized in all cases is much lower. In the latter case, the total activity displayed by the hydrophilized beads is comparable to that obtained for the unmodified beads, while for the other polymers, the resulting total activity is lower than that for the unmodified beads.
3.3. Immobilization from microemulsion at hydrophilized beads As discussed above, the protein rejecting prop-
erties of uncharged hydrophilic polymer coatings are well known and due to a combination of a steric repulsive interaction between the polymer layer and the protein, and the lack of strong electrostatic, van der Waals and other attractive interactions [1]. However, the steric interaction depends strongly on the solvency of the polymer [1,48–50]. Therefore, while uncharged flexible polymers are efficient protein rejectants at good solvency conditions, their performance deteriorate at poor solvency conditions due to a contraction of the interfacial layer. With this in mind, several different immobilization techniques based on good-poor solvency cycles have been developed [9,10]. One of these is based on a water-in-oil microemulsion [9]. In such a microemulsion, the protein is present in water droplets dispersed in the oil continuous phase, while the polymer layer experiences poor solvency conditions due to contact with the non-aqueous phase. Therefore, immobilization could be expected to be better from such a microemulsion than from aqueous solution, at least provided that the protein activity is not lost by the contact with surface active agents present in the microemulsion. Indeed, it has been shown before that such a procedure results in a higher immobilization yield than immobilization from aqueous solution [9]. Considering this, we investigated the immobilization of trypsin from such a microemulsion to GMA-GDMA beads hydrophilized through various polymer coatings. As can be seen from Table 2, a significant immobilization yield is obtained for all the coatings, and for all coatings a significantly higher total activity was obtained compared to the unmodified beads. The total activity increase compared to immobilization at the hydrophilized beads from
Table 1 Immobilization of trypsin at hydrophilized GMA-GDMA beads from 0.3 M borate buffer, pH 8. Trypsin concentration 11.8 mg/ml. The specific activity of trypsin in aqueous solution was 50 95 BAEE/mg trypsin Coating
Activity (BAEE/g bead)
Amount (mg/g bead)
Specific activity (BAEE/mg trypsin)
Unmodified HEC PVA 88% Dextran T40
145 64 78 119
40.2 8.4 9.7 8.1
3.6 7.7 8.0 14.6
M. Malmsten, A. Larsson / Colloids and Surfaces B: Biointerfaces 18 (2000) 277–284
283
Table 2 Activity of trypsin immobilized at various hydrophilized GMA-GDMA beads by the microemulsion procedure. Trypsin concentration 11.8 mg/ml. The specific activity of trypsin in aqueous solution was 50 9 5 BAEE/mg trypsin Coating
Activity (BAEE/g bead)
Amount (mg/g bead)
Specific activity (BAEE/mg trypsin)
Unmodified Phenyldextran Dextran AB Dextran T40 HEC PVA 80% PVA 88% PEI-PEO
159 174 229 249 245 207 199 224
32.4 24.0 23.7 23.8 26.1 24.3 29.4 29.7
4.9 7.3 9.5 10.5 9.4 8.5 6.8 7.5
aqueous solution is due to an increased immobilization yield, whereas the total activity improvement compared to the unmodified beads is due to an increased specific activity. Although the results in Table 2 display some variation between the different polymer coatings, which are presently poorly understood and require further studies, the main point is that quailitatively similar results are obtained for a rather wide range of hydrophilic polymer coatings, underlining the generality of the approach. In previous investigations, protein immobilization at hydrophilized surfaces from a microemulsion has been utilized in several contexts. For example, Thomas et al., and Malmsten et al. investigated the immobilization of antibodies in relation to solid phase diagnostics, whereas Moachon et al. investigated the usefulnes of such coatings in extracorporeal therapy [1,5,6]. In these and other studies, the approach taken has shown very low levels of unspecific adsorption, high signal-to-noice-ratio, and superior performance in different biomedical applications. Also the solvency approach has been used before to improve the immobilization yield at hydrophilized surfaces, using either temperature [10] or microemulsion [9] to deteriorate the solvency conditions for the hydrophilic polymer coatings. However, to our knowledge this is the first time that the usefulnes of the approach taken also for bioprocessing is illustrated. Examples of such applications where this immobilization procedure holds promise include manufacturing of protein hydrolysates for reducing allergic problems with childrens dairy
products, self-cleaning ultrafiltration units, and wound cleaning compression textiles, just to mention a few.
4. Summary The immobilization of trypsin at porous glycidyl methacrylate (GMA-GDMA) beads was investigated regarding immobilization yield and retained activity after immobilization. In particular, the immobilization at unmodified GMAGDMA beads was compared to that at beads hydrophilized by various polymer coatings, as was the immobilization at these beads from aqueous solution and a water-in-oil microemulsion. While the immobilization yield was lower for the hydrophilized than for the unmodified beads, the retained specific activity was larger. By performing the immobilization at hydrophilized beads from the microemulsion, a higher yield than obtained by immobilization at hydrophilized beads from aqueous solution could be combined with a higher specific activity than obtained by immobilization at the unmodified beads, thereby resulting in improved total activities. At the same time, however, the (water-in-oil) microemulsion stability area may, as in the present case, limit the protein concentration, which can be used in the immobilization procedure, particularly at high electrolyte concentration. This emphasizes the need in future work to identify microemulsions with a larger protein solubilization capacity.
284
M. Malmsten, A. Larsson / Colloids and Surfaces B: Biointerfaces 18 (2000) 277–284
Acknowledgements Mats Gruvega˚rd is thanked for bead preparation and analysis, while Eva Lundgren is acknowledged for performing the experimental bead functionalization work. Furthermore, the authors would like to thank Mats Gruvega˚rd and PerMikael A, berg, Amersham Pharmacia Biotech, for fruitful discussion. This study was part of the Brite Euram project ‘Solid Matrices for Bioprocesses’ (BE95-1075), and was funded by EU and the Swedish National Board for Industrial and Technical Development (NUTEK).
References [1] M. Malmsten (Eds.), Biopolymers at Interfaces, Surfactant Science Series, vol. 75, Marcel Dekker, New York, 1998. [2] M. Malmsten, J. Colloid Interface Sci. 207 (1998) 186. [3] A. Kondo, F. Murakami, M. Kawagoe, K. Higashitani, Appl. Microbiol. Biotechnol. 39 (1993) 726. [4] J.M. Harris, Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications, Plenum Press, New York, 1992. [5] M. Malmsten, B. Lassen, K. Holmberg, V. Thomas, G. Quash, J. Colloid Interface Sci. 177 (1996) 70. [6] V. Thomas, K. Bergstro¨m, G. Quash, K. Holmberg, Colloid Surf. A 77 (1993) 125. [7] J.M. Harris, K. Yoshinaga, J. Bioact. Compat. Polym. 4 (1989) 281. [8] M.B. Stark, K. Holmberg, Biotech. Bioeng. 34 (1989) 942. [9] K. Bergstro¨m, K. Holmberg, Colloids Surf. 63 (1992) 273. [10] F. Tiberg, C. Brink, M. Hellsten, K. Holmberg, Colloid Polym. Sci. 270 (1992) 1188. [11] M. Malmsten, B. Lindman, Langmuir 6 (1990) 357. [12] D. Bharadwaj, A. Mukherjee, M.S. Roy, R.N. Hati, R.N. Mukherjee, Ind. J. Biochem. Biophys. 29 (1992) 375. [13] P.C. Lorenzen, E. Schlimme, Z. Erna¨rungswiss. 34 (1995) 118. [14] R. Batra, R. Tyagia, M.N. Gupta, Biocatal. Biotransform. 15 (1997) 101. [15] M.A. Cohenford, P.F. Santoro, J.C. Urbanowski, J.A. Dain, Biotechnol. Bioeng. 28 (1986) 736. [16] A. Kumar, M.N. Gupta, J. Mol. Catal. B 5 (1998) 289. [17] H.A. Chase, Y. Yang, Biotechnol. Appl. Biochem. 27 (1998) 205. [18] P. Wang, T.G. Hill, C.A. Wartchow, et al., J. Am. Chem. Soc. 114 (1992) 378. [19] E.A. Kulik, K. Kato, M.I. Ivanchenko, Y. Ikada, Biomaterials 14 (1993) 763.
[20] M. Alves Da Silva, M.H. Gil, J. Guiomar, et al., Radiat. Phys. Chem. 36 (1990) 589. [21] S. Devi, J.T. Guthrie, C.G. Beddows, Radiat. Phys. Chem. 36 (1990) 697. [22] D. Lewis, T.L. Whateley, Biomaterials 9 (1988) 71. [23] D. Wu, R.R. Walters, J. Chromatogr. 458 (1988) 169. [24] S. Kamei, M. Okubo, T. Matsumoto, J. Appl. Polym. Sci. 34 (1987) 1439. [25] M.A. Jasim, G.M. Hall, J. Mann, K.D.A. Taylor, J. Chem. Tech. Biotechnol. 40 (1987) 251. [26] J. Bo, X. Ming, Eur. Polym. J. 28 (1992) 827. [27] R.M. Blanco, J.M. Guisan, Enzyme Microb. Technol. 11 (1989) 360. [28] R.M. Blanco, J.J. Calvete, J.M. Guisan, Enzyme Microb. Technol. 11 (1989) 353. [29] H. Dauzenberg, N. Karibyants, S.Y. Zaitsev, Macromol. Rapid Commun. 18 (1997) 175. [30] S. Kamei, M. Okubo, T. Matsuda, T. Matsumoto, Colloid Polym. Sci. 264 (1986) 743. [31] T. Shiroya, N. Tamura, M. Yasui, K. Fujimoto, H. Kawaguchi, Colloids Surf. B 4 (1995) 267. [32] M. Yasui, T. Shiroya, K. Fujimoto, H. Kawaguchi, Colloids Surf. B 8 (1997) 311. [33] E.T. Kang, K.L. Tan, K. Kato, Y. Uyama, Y. Ikada, Macromolecules 29 (1996) 6872. [34] S. Devi, J.T. Guthrie, C.G. Beddows, Radiat. Phys. Chem. 36 (1990) 703. [35] M. Okubo, H. Ahmad, J. Polym. Sci. A 36 (1998) 883. [36] C.G. Beddows, M.H. Gil, J.T. Guthrie, J. Appl. Polym. Sci. 35 (1988) 135. [37] M. Rucka, G. Pozniak, B. Turkiewicz, W. Trochimczuk, Enzyme Microb. Technol. 18 (477) (1996) 477. [38] M. Sardar, R. Agarwal, A. Kumar, M.N. Gupta, Enzyme Microb. Technol. 20 (1997) 361. [39] P.C. Lorenzen, H. Fischer, E. Schlimme, Die Nahrung 38 (1994) 551. [40] J. Turkova, K. Blaha, M. Malanikova, D. Vancurova, F. Svec, J. Kalal, Biochim. Biophys. Acta 524 (1978) 162. [41] J. Porath, N. Fornstedt, J. Chromatogr. 51 (1970) 479. [42] I. Matsumoto, Y. Mizuno, N. Seno, J. Biochem. 85 (1979) 1091. [43] L. Sundberg, J. Porath, J. Chromatogr. 90 (1974) 87. [44] S. Avramiotis, P. Lianos, A. Xenakis, Biocatal. Biotransform. 14 (1997) 299. [45] I.N. Chang, J.N. Herron, Langmuir 11 (1995) 2083. [46] C.S. Lee, G. Belfort, Proc. Natl. Acad. Sci. USA 86 (1989) 8392. [47] M. Malmsten, K. Emoto, J.M. Van Alstine, J. Colloid Interface Sci. 202 (1998) 507. [48] J.N. Israelachvili, Intermolecular Surface Forces, Academic Press, London, 1992. [49] G.J. Fleer, M.A. Cohen Stuart, J.M.H.M. Scheutjens, T. Cosgrove, B. Vincent, Polymers at Interfaces, Chapman and Hall, London, 1993. [50] D.H. Napper, Polymeric Stabilization of Colloidal Dispersions, Academic Press, London, 1983.