Journal of Membrane Science 181 (2001) 29–37
Catalytic biofunctional membranes containing site-specifically immobilized enzyme arrays: a review D. Allan Butterfield a,c,∗ , Dibakar Bhattacharyya b,c , Sylvia Daunert a,c , Leonidas Bachas a,c a
c
Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055, USA b Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY 40506-0046, USA Center of Membrane Sciences, University of Kentucky, Lexington, KY 40506-0057, USA
Received 22 October 1999; received in revised form 14 January 2000; accepted 18 January 2000
Abstract Biofunctional membranes normally involve the random immobilization of biomolecules to porous, polymeric membranes, often through the numerous lysine residues on the protein. In this process, bioactivity is significantly decreased largely due to different orientations of the biomolecule with respect to the membrane or to multiple point attachment. To circumvent this difficulty, while still taking advantage of the immobilization of biomolecules, site-specific immobilization of the biomolecule with the active (or binding) site directed away from the membrane is essential. In this review, we summarize our efforts involving biophysical and bioanalytical chemistry and chemical engineering, together with molecular biology, to develop and characterize such site-specifically membrane immobilized catalytic enzyme bioreactors. Site-directed mutagenesis, gene fusion technology, and post-translational modification methods are employed to effectuate the site-specific membrane immobilization. Electron paramagnetic resonance, in conjunction with active-site specific spin labels, kinetic analyses, and membrane properties are used to characterize these systems. Biofunctional membranes incorporating site-specifically immobilized biomolecules provide greater efficiency of biocatalysis, bioseparations, and bioanalysis. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Biofunctional membranes; Membrane-bound enzymes; Site-specific immobilization using molecular biology; Membrane reactors
1. Introduction Traditional membrane technology has provided considerable insight into separation and reaction. However, many researchers now agree that new advances in membrane technology will require synergy between the strength and processing properties of ∗ Corresponding author. Tel.: +1-606-257-5875; fax: +1-606-257-5876. E-mail address:
[email protected] (D.A. Butterfield).
traditional polymeric membranes and the selectivity through molecular recognition of biological membranes, so-called, biofunctional membranes. Biofunctional membranes are entities in which a biomolecule, collection of biomolecules or cells are immobilized onto polymeric matrices cast in the form of porous membranes [1]. Biofunctional membranes have been used in catalysis (membrane-based enzyme bioreactors), separations (affinity membranes), analysis (biosensors; metal ion-specific separations), and artificial organs. These uses of biofunctional membranes
0376-7388/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 6 - 7 3 8 8 ( 0 0 ) 0 0 3 4 2 - 2
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Fig. 1. (A) Random immobilization of enzymes, showing potential difficulties. (B) Site-specific immobilization of enzymes to form an array of similarly oriented proteins, always with the active site away from the polymer surface. See text.
take advantage of molecular recognition chemistry, prominent in biological membranes. Although immobilization of enzymes generally enhance their stability [1], one major disadvantage of random immobilization of enzymes onto polymeric microfiltration type membranes is that the activity of the immobilized enzyme is often significantly decreased because the active site may be blocked from substrate accessibility, multiple point-binding may occur, or the enzyme may be denatured [2–7] (Fig. 1). The extent of decrease in enzyme activity is also a strong function of the membrane hydrophobicity: the randomly immobilized enzyme activity follow the following order: pure cellulose membrane (cellulose acetate) polysulfone-based membrane. In particular, traditional enzyme immobilization is often accomplished as shown in Fig. 1, in which the enzyme is randomly immobilized either directly on the membrane or via a spacer arm, often through the ε-amino functionality of lysine residues on the protein. Because the protein often contains multiple lysine residues spread over the surface of the enzyme, different orientations of the enzyme with respect to the membrane occur, some completely blocking the active site from interaction with substrate. In addition, multiple point attachment could occur [1], and with random immobilization high enzyme loading is not possible [13]. Site-specific immobilization using the power of molecular biology can overcome some of these difficulties. For example, we and others have demonstrated that enzymes can be immobilized in an ordered and site-specific manner on beads and membranes to form enzyme arrays, and that these enzymes have superior catalytic properties compared to enzymes that have been immobilized by using conventional approaches
[8–22]. Three different approaches to site selective immobilization of enzymes have been explored: (a) Gene fusion to incorporate a peptidic affinity tag at the N- or C-terminus of the enzyme. The enzymes are then attached from this affinity tag to anti-tag antibodies on membranes; (b) post-translational modification to incorporate a single biotin moiety on enzymes. The enzymes can be attached to membranes through a (strept) avidin bridge; (c) site-directed mutagenesis to introduce unique cysteines to enzymes. The enzymes are attached on thiol-reactive surfaces through the sulfhydryl group on the side chain of the introduced cysteine, which is located on the opposite side of the protein from the active site. In all these methods, the active sites of the immobilized enzymes face away from the polymeric surface and a consequent higher activity results. Membrane immobilization also provides added benefits in terms of temperature stability and minimization of product inhibition [1–29]. These different approaches were developed in order to accommodate site-specific immobilization of enzymes with different structural characteristics. If, for example, an enzyme does not contain any cysteines (or any cysteines that are required for activity), then the site-directed mutagenesis approach may be used. The gene fusion approach is appropriate when the N- or C-terminus of the enzyme are away from the active site, and the post-translational modification incorporates a biotin moiety on a specific lysine residue on the enzyme, which allows the use of an avidin spacer between the enzyme and the immobilization surface. The specific examples that we review herein provide insight into how the catalytic properties of immobilized enzymes can be enhanced. In addition, electron paramagnetic resonance (EPR), in conjunction with active site-specific spin labels, monitors the conformation of the active site of the enzyme. Denaturation in solution or random immobilization to polymeric membranes leads to drastic changes in active site conformation [2–4,10,30–37]. EPR studies, in conjunction with site-specific immobilization, showed that the active site structure of these immobilized enzymes resemble more closely that of enzymes in solution than that of randomly immobilized enzymes, and this enhancement of structure may be related to the enhancement of enzyme performance in these site-specifically immobilized systems. This
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review summarizes our integrated, multidisciplinary studies of catalytic biofunctional membrane enzyme arrays.
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which the paramagnetic center of the spin label is found (reviewed in [30,31,35]). 2.3. Molecular biology techniques employed
2. Methods 2.1. Membrane considerations The choice of membrane support for enzyme immobilization is governed by several factors, among which are the relative hydrophilicity/hydrophobicity of the polymer from which the membrane is formed, the absence of micropores below 0.05 m, and the ease of functionalization to provide binding sites for the enzymes [1,39]. The key is to minimize the hydrophobicity of the membrane, which can often lead to non-specific enzyme binding to the polymer surface, i.e. lack of binding specificity and enhanced probability for enzyme denaturation can result [1]. Commercial membranes with aldehydic or other reactive group functionality are available, and these are usually cellulosic-, silica-, or poly(ether sulfone)-based membranes. The results of studies from our laboratory reviewed here mostly utilized modified poly(ether sulfone) [MPS] or silica-based membranes. The MPS membranes, containing a polyaldehyde layer, have 9.1 m2 /g BET surface area and an average pore diameter of 0.45 m [2–4,10]. In general, one can also prepare membranes from cellulose-based materials [29]. For example, cellulose acetate can be easily hydrolyzed to cellulose by alkali treatment, and subsequent oxidation by periodate leads to aldehyde/ functionalized membranes [38]. By varying functionalization and casting conditions, such membranes can be selectively prepared with varying degrees of aldehyde incorporation, pore size, and thickness [38]. 2.2. EPR spin labeling studies Spin labeling is a paramagnetic resonance method in which a stable nitroxide molecule is attached to the system of interest and the EPR spectrum obtained. The power of the technique rests in the extreme sensitivity of EPR, the relative simplicity of the nitroxide spectra that need to be analyzed, and the significant information available on molecular motion, polarity, and interactions of the local microenvironment in
Approaches to site-specific immobilization require functionalization of the target molecule, tailoring the surface, or both. Most literature procedures used for immobilization of proteins are reactions where covalent bonds are formed between surface-exposed functional groups (typically, amino or carboxyl) of amino acids or generated aldehydes on proteins, with suitably modified solid supports, membrane surfaces or molecular aggregates. These reactions typically lead to multiple bond formation between the immobilization surface and more than one amino acid residue on the same protein molecule (Fig. 1). As discussed above, this can affect the biological activity of the immobilized protein. Recombinant DNA techniques are used in our laboratories to introduce specific sites of attachment on a particular protein. The most useful techniques in that regard include site-directed mutagenesis to introduce unique amino acid residues (e.g. a single cysteine) and gene modifications that introduce a sequence of amino acids that serve as an affinity site for immobilization. Site-directed mutagenesis can be used to introduce a free cysteine at a suitable position on the protein, which can be subsequently used for immobilization. Fusion protein technology can be employed to introduce a recognition group on the N- or C-terminus of the enzyme. For example, biotin or FLAG can be introduced to the enzyme, and site-specific immobilization via (strept) avidin or anti-FLAG antibody and protein A can ensue [8–13]. 2.4. Enzyme immobilization methods Random covalent immobilization of enzymes normally takes advantage of the reaction of lysine — amino groups of enzymes with aldehyde moieties on the membrane [1,39,40]. Other reactions are possible. The outcome depends on the nature of the membrane support, e.g., hydrophilicity, hydrophobicity, reactive functional group density, porosity, pore-size distribution, etc. [1]. Our membrane studies have included modified polysulfone, cellulosics, and composite silica materials.
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3. Results and discussion 3.1. Random immobilization of enzymes on membranes: structure and activity EPR, in conjunction with appropriate spin labels, permits selective spin labeling of the active sites of enzymes [35]. For example, the active-site sulfhydryl group of papain can be spin labeled with the methane thiosulfonate spin label, MTS [2–4,10,11] and the serine active site of subtilisin can be specifically spin labeled with 4-ethoxy (fluorophosphinyloxy)-Tempo (4-EFT) [37]. Molecular motion of the spin label in the active site can be assessed in several ways, but perhaps the most sensitive and easiest for in-phase EPR is the measurement of the outer hyperfine ex0 . As motion slows due to denaturation, trema, 2Azz this parameter increases because of increased dipolar hyperfine interactions. Immobilization of spin labeled enzymes leads to a spectrum with two environments, A and D, respectively, representative of active and denatured environments [2,10], similar to observations of other enzymes immobilized on beads (reviewed, [35]). Spin labeling of previously randomly immobilized papain or modeling studies [2,4], showed only the fraction of the population of enzymes with active enzyme could be labeled, and this fraction was approximately 25%. Enzymes can be immobilized and spin labeled on hollow fiber membranes as well as flat sheet supports [3]. The denaturation parameter, I(D)/I(A) [2], where I refers to the EPR signal amplitude of the MI =0 central line below the baseline [2], is a sensitive parameter to measure the relative contributions of the active and denatured population of immobilized enzymes to the EPR signal. As expected, I(D)/I(A) increases in the presence of denaturants [2]. We have used in-phase EPR to learn much about immobilized enzymes [1,11,13], the main conclusions of which can be summarized as follows: (a) Generally, random immobilization of enzymes leads to a closing of the active site cleft, reflective of decreased motion of the active-site bound spin label; (b) The effects of denaturants, temperature, pH, storage stability, reusability, etc., can all be assessed using EPR spin labeling, and correlates well with the activity and increased stability associated with immobilization; (c) Immobilization of enzymes using site-specific methods described below generally leads to faster
motion of the active site-bound spin label, and this is correlated with higher activity than is the case for randomly-immobilized enzymes, i.e. motion closer to that of the respective solution enzyme. 3.2. Site-specific immobilization of enzymes in conjunction with molecular biological methods As noted, random immobilization of enzymes often leads to structural deformations, to inability to predict and control structure/activity relationships, and to reduced catalytic activity. In order to circumvent these difficulties, production of a highly ordered, two-dimensional array of enzymes on membranes is desired (Fig. 1). This alignment has the distinct advantage of orienting the active site away from the polymeric surface, thereby giving the substrate nearly full accessibility. However, by having the enzyme immobilized, greater resistance to denaturants, pH, and temperature is produced [1]. Protein orientation in biological membranes is a well-known phenomenon. For example, transmembrane protein channels and pumps are aligned in a specific manner to permit passive, facilitated, or active transport of important moieties across cell membranes [39]. Biofunctional membranes are designed to incorporate biological properties to synthetic matrices, so orientation of proteins is just one more way in which the function of this class of membranes can be enhanced. There are several approaches to oriented immobilization of enzymes [40], including (a) binding of site-specifically biotinylated enzymes to membraneimmobilized (strept)avidin; (b) fusion protein technology coupled with affinity tags on membranes; and (c) site-directed mutagenesis to introduce a cysteine into a protein with subsequent SH-specific coupling to the membrane support. Each of these will be discussed. Biotin, a vitamin, has an affinity for avidin, a protein found in egg-whites that is remarkably strong, rivaling that of a covalent bond, even though the two moieties form a non-covalent association. The Kd of the avidin–biotin complex is approximately 10−15 . Molecular biology can be used to accomplish biotin addition in a site-specific manner. For example, we showed that β-galactosidase (β-Gal) could be biotinylated at a single site following expression of a plasmid in E. coli that encodes a fusion protein of β-Gal and a polypeptide tag at its N-terminus. When this fusion
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Fig. 2. Site-specific immobilization of enzymes, in this case β-galactosidase, using molecular biology. A biotin ligase recognition sequence is added to the DNA of β-galactosidase in the plasmid that is incorporated into the E. coli. Subsequent expression and purification of the β-galactosidase containing a N-terminal domain biotin allows this complex to be added specifically to (strept)avidin previously immobilized onto the membrane. See text.
product is expressed in E. coli, one lysine residue on the polypeptide tag is specifically biotinylated by the bacteria-resident enzyme, biotin ligase, during the post-translational modification process (Fig. 2). Subsequent isolation and purification of this product was followed by addition to poly(ether sulfone) membranes onto which avidin was already immobilized. A two-fold improvement in enzyme activity was observed relative to that of fully biotinylated enzyme, and a nearly 20-fold enhancement of activity was found compared to randomly immobilized β-galactosidase [8]. Different immobilization strategies, coupled with gene fusion methods, were employed to investigate site-specifically immobilized alkaline phosphatase (AP) [9]. In this approach a fusion protein of AP and FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) is produced and purified by affinity methods. The fusion protein is reacted with a monoclonal antibody (mAb) directed against FLAG. Protein A, which has a high binding towards and specificity for monoclonal IgG anti-FLAG antibody, is immobilized on the membrane (Fig. 3). Subsequent reaction leads to highly specific molecular recognition chemistry taking place with resulting immobilization of alkaline phosphatase in a site-specific orientation [9]. Site-specific immobilization of AP resulted in a higher catalytic activity relative to the randomly immobilized enzyme, and
this improvement reached to over 85% of the activity of the soluble enzyme [9]. Site directed mutagenesis was used to introduce a cysteine into subtilisin [12,13], a protein normally devoid of cysteines. Subtilisin is a serine protease,
Fig. 3. Site-specific immobilization of enzymes using fusion protein technology. The DNA sequence specific for the octapeptide FLAG is added to that of the enzyme of interest at the N- or C-terminus. After expression and purification, this complex is added to membrane. On the membrane, protein A, which has a high affinity for IgG antibodies, is immobilized, followed by the mAb to FLAG. Addition of dimethyl pimelimidate (DMP) stabilizes the anti-FLAG-protein A complex. Addition of the FLAG-enzyme fusion complex results in a specific orientation of the enzyme with the active site away from the surface.
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Table 1 Characteristics of subtilisin with serine to cysteine mutations Subtilisin BPN’
KM (mM)
kCAT (s−1 )
Cysteine content
Wild type Subtilisin (S249C) Subtilisin (S145C)
0.16±0.01 0.19±0.01 0.21±0.02
51.3±1.6 45.7±1.5 48.5±2.4
0 1.06 0.97
with additional serine residues on the distal side of the protein away from the active site serine. Site-directed mutagenesis was used to introduce a cysteine, one at a time, in these distal serines. Cysteine is a conservative mutation for serine, replacing the OH functionality by SH. Analysis for cysteine content demonstrated that only one cysteine was introduced into the protease (Table 1). Kinetic analysis of the mutated enzyme in solution showed that no significant difference in activity was found, and EPR studies indicated that there was no change in mobility of a spin label bound in the active site. All these findings suggested that the mutations of Ser by Cys were innocuous to the structure and function of subtilisin. Site-specific immobilization of enzymes can be accomplished using site-directed mutagenesis. Employing the unique cysteine located away from the active site, the SH group is reacted with a thiol-reactive membrane support (Fig. 4). Using this methodology, an increase in relative activity from 48 to 83% compared to that of the soluble enzyme, was observed for randomly immobilized and site-specifically immobilized subtilisin on PVC-silica membranes. EPR showed increased motion of the active site in the latter compared to the former [13].
Fig. 4. Site-specific immobilization of enzymes using site-directed mutagenesis. To a protein with no cysteines, this amino acid is added in lieu of serine at a site distal from the active site of the enzyme. Subsequent reaction of the enzyme with the surface containing a SH reactive moeity leads to site-specific immobilization of the enzyme. See text.
Fig. 5. A rechargeable site-system of site-specific membrane immobilization of enzymes. Phenothiazine is immobilized to the membrane support, and the fusion protein complex of calmodulin and enzyme is added to the modified support in the presence of Ca2+ . This permits a hydrophobic pocket in calmodulin to bind to the phenothiazine. When the enzyme is spent, EGTA, a specific Ca2+ chelator, is added to remove the Ca2+ from the calmodulin. This causes the protein to fold, dissociating itself from the phenothiazine. To recharge the system, a new calmodulin-enzyme complex is added in the presence of Ca2+ . See text.
In some applications of biocatalytic membrane reactors, it would be desirable to recharge spent systems. Using our fusion protein technology, this is now quite easily accomplished (Fig. 5). The enzyme and calmodulin, a Ca2+ -binding protein with high affinity for phenothiazine in the presence of Ca2+ , are produced as a fusion protein complex by recombinant DNA methods [41]. After expression and purification, the complex is added to the cellulosic membrane support in the presence of Ca2+ , effecting a site-specific immobilization. When the enzyme is spent, one simply adds EGTA, a specific Ca2+ chelator to the system, resulting in a rapid and complete dissociation of calmodulin from phenothiazine. A new calmodulin-enzyme fusion protein is added in the presence of Ca2+ to recharge the system. This approach has a particular advantage when the support cost is high; this method allows expensive supports to be reused. Enzyme activity is a function of enzyme loading in catalytic biofunctional membranes [4]. Sitespecifically immobilized enzyme arrays have a much higher activity per mg bound enzyme relative to randomly immobilized enzyme (Fig. 6) [13]. Thus, the activity of randomly immobilized enzymes saturates at a low activity or at low level of immobilized enzyme compared to site-specifically immobilized enzymes. Reduced steric hindrance of the active site in enzyme arrays by adjacent enzyme molecules and by the mem-
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Fig. 6. Effect of enzyme loading on the activity of subtilisin immobilized onto modified poly(ether sulfone) membranes randomly (circles) or following site-directed mutagenesis-based site-specific immobilization.
brane surface is the result of each enzyme molecule being oriented in the same manner with the active site away from the polymeric membrane surface (Fig. 1). This orientation provides the clear advantages over randomly immobilized enzymes: higher activity (in some cases, approaching that of the soluble enzyme), higher loading, and an active-site structure similar to that of the soluble enzyme. It should be noted that even with highly active site-directed immobilized enzymes, the use of convective flow in membranes eliminates substrate diffusion problems. As a test of these ideas, namely that oriented enzymes with the active site directed away from the polymer surface should show greater activity, we measured the trans-esterification reaction catalyzed by subtilisin in an organic solvent. Using 1-butanol and CBZ-alanine-p-nitrophenyl ester, the rate of the reaction in water-saturated hexane was determined for randomly immobilized and site-specifically immobilized subtilisin that had been mutated to introduce a cysteine in a location distal from the active site. This was a stringent test of our notion, since subtilisin’s protease activity is higher than its esterase activity, and since the reaction occurs in organic media, even lower rate would be expected. However, consistent with our hypothesis, greater than two-fold enhancement of the rate was found for subtilisin that had been subjected
to site-directed mutagenesis and site-specifically immobilized onto PVC-silica membranes [13]. Studies using saturation-transfer EPR methods to enable slow motion to be investigated were consistent with the activity measurements [13].
4. Conclusions and future studies The power of the studies reviewed here lies in their multidisciplinary and synergistic approaches. Biophysical chemistry, bioanalytical chemistry, molecular biology, and chemical engineering techniques are combined to gain insights into these biofunctional membranes. Further investigation of these site-specifically immobilized enzymes, binding proteins, and other biological molecules to study catalysis, analysis, and separation is envisaged to produce greater fundamental understanding and application of catalytic biofunctional membrane enzyme arrays, biosensors, and affinity membranes. Directed evolution has been used recently to enhance the catalytic properties and stability of enzymes in solution. One may envisage that enzymes obtained through directed evolution could also be site-selectively immobilized on proteins by following one of the approaches outlined in this review to further enhance the properties of
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biofunctional membranes. EPR, both in-phase and saturation transfer, has predictive power for biofunctional membrane effectiveness, and the development of functionalized solvent resistant membranes along with the use of non-aqueous solvents could also allow enhancement of biocatalytic reaction selectivity. Such studies are ongoing in our Center of Membrane Sciences.
Acknowledgements This work was supported in part by grants from NSF (CTS-9307518) and DoD (DAAG55-98-1-0003). We thank J. Wang for assistance in preparation of some of the figures in this review.
References [1] D.A. Butterfield, Biofunctional Membranes, Plenum Press, New York, 1996. [2] D.A. Butterfield, J. Lee, S. Ganapathi, D. Bhattacharyya, Biofunctional membranes IV. Active site structure and stability of an immobilized enzyme, papain, on modified polysulfone membranes studies by electron paramagnetic resonance and kinetics, J. Membr. Sci. 91 (1994) 47. [3] S. Ganapathi, D.A. Butterfield, D. Bhattacharyya, Flat sheet and hollow fiber membrane bioreactors: a study of the kinetics and active site conformational changes of immobilized papain including sorption studies of reaction constituents, J. Chem. Technol. Biotechnol. 64 (1995) 157. [4] S. Ganapathi, D.A. Butterfield, D. Bhattacharyya, Kinetics and active fraction determination of a protease enzyme immobilized on functionalized membranes: mathematical modeling and experimental results, Biotechnol. Prog. 14 (1998) 865. [5] P. Zhuang, D.A. Butterfield, Optimization of covalently coupling enzymes to polymeric membranes: EPR studies of papain, J. Appl. Polym. Sci. 47 (1993) 1329. [6] P. Zhuang, D.A. Butterfield, Spin labeling and kinetic studies of a membrane-immobilized proteolytic enzyme, Biotechnol. Prog. 8 (1992) 204. [7] P. Zhuang, D.A. Butterfield, Structural and enzymatic characterizations of papain immobilized onto vinyl alcohol/vinyl butyral copolymer membrane, J. Membr. Sci. 66 (1992) 247. [8] S. Vishwanath, D. Bhattacharyya, W. Huang, L.G. Bachas, Site-directed and random enzyme immobilization on functionalized membranes: kinetic studies and models, J. Membr. Sci. 108 (1995) 1. [9] S.K. Vishwanath, C.R. Watson, W. Huang, L.G. Bachas, D. Bhattacharyya, Kinetic studies of site-specifically and randomly immobilized alkaline phosphatase on functionalized membranes, J. Chem. Technol. Biotechnol. 68 (1997) 294.
[10] A. Bhardwaj, J. Lee, S. Glauner, S. Ganapathi, D. Bhattacharyya, D.A. Butterfield, Biofunctional Membranes: an EPR study of active site structure and stability of papain non-covalently immobilized on the surface of modified poly (ether) sulfone membranes through the avidin-biotin linkage, J. Membr. Sci. 119 (1996) 241. [11] D.A. Butterfield, R. Subramaniam, D. Bhattacharyya, S. Viswanath, W. Huang, L. Bachas, Biofunctional membranes: electron paramagnetic resonance studies of the active site structure of enzymes site-specifically immobilized onto polymeric supports through molecular recognition, Polym. Mat. Sci. Eng. Preprints 76 (1997) 602. [12] W. Huang, J. Wang, D. Bhattacharyya, L.G. Bachas, Improved enzymatic activity by site-specific immobilization of subtilisin, Anal. Chem. 69 (1997) 4601. [13] S. Vishwanath, J. Wang, L.G. Bachas, D.A. Butterfield, D. Bhattacharyya, Site-directed and random immobilization of subtilisin on functionalized membranes: activity determination in aqueous and organic media, Biotech. Bioeng. 60 (1998) 608. [14] S.V. Rao, K.W. Anderson, L.G. Bachas, Oriented immobilization of proteins, Mikrochim. Acta 128 (1998) 127. [15] M. Persson, L. Bülow, K. Mosbach, Purification and sitespecific immobilization of genetically engineered glucose dehydrogenase on thiopropyl-sepharose, FEBS Lett. 270 (1990) 41. [16] H.K.W. Kallwass, W. Parris, E.L.A. McFarlane, M. Gold, J.B. Jones, Site-specific immobilization of an l-lactate dehydrogenase via an engineered surface cysteine residue, Biotechnol. Lett. 15 (1993) 29. [17] S.J. Vigmond, M. Iwakura, F. Mizutani, T. Katsura, Site-specific immobilization of molecularly engineered dihydrofolate-reductase to gold surfaces, Langmuir 10 (1994) 2860. [18] M.A. McLean, P.S. Stayton, S.G. Sligar, Engineering protein orientation at surfaces to control marcromolecular recognition events, Anal. Chem. 65 (1993) 2676. [19] A. Chilkoti, G. Chen, P.S. Stayton, A. Hoffman, Site-specific conjugation of a temperature-sensitive polymer to a genetically-engineered protein, Bioconjugate Chem. 5 (1994) 504. [20] E. Hochuli, W. Bannwarth, H. Döbeli, R. Gentz, D. Stüber, Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent, Bio/Technology 6 (1988) 1321. [21] J. Carlsson, K. Mosbach, L. Bülow, Affinity precipitation and site-specific immobilization of proteins carrying polyhistidine tails, Biotechnol. Bioeng. 51 (1996) 221. [22] E. Ong, N.R. Gilkes, A.J. Warren, R.C. Miller Jr., D.G. Kilburn, Enzyme immobilization using the cellulose-binding domain of a Cellulomonas fimi exoglucanase, Bio/Technology 7 (1989) 604. [23] H.R. Allcock, S. Kwon, Covalent linkage of proteins to surface-modified poly (organophosphazenes): immobilization of glucose-6-phosphate dehydrogenase and trypsin, Macromolecules 19 (1986) 1502. [24] A.P. Bakken, C.G. Hill Jr., C.H. Amundson, Hydrolysis of lactose in skin milk by immobilized β-galactosidase in a spiral flow reactor, Biotech. Bioeng. 33 (1989) 1249.
D.A. Butterfield et al. / Journal of Membrane Science 181 (2001) 29–37 [25] P.L. Domen, J.R. Nevens, A.K. Mallia, G.T. Hermanson, D.C. Klenk, Site-directed immobilization of proteins, J. Chromatogr. 510 (1990) 293. [26] T.M. Spitznagel, J.W. Jacobs, D.S. Clark, Random and site-specific immobilization of catalytic antibodies, Enzyme Microb. Technol. 15 (1993) 916. [27] E. Klein, Affinity Membranes, Wiley, New York, 1990, p. 129. [28] G.A. Marg, G.L. Milhauser, P.S. Skerker, D.S. Clark, Application of EPR methods in studies of immobilized enzymes systems, Ann. N.Y. Acad. Sci. 469 (1986) 253. [29] R. Ulbricht, A. Schellenberger, Studies on thermal inactivation of immobilized enzymes, Biotech. Bioeng. 28 (1986) 511. [30] D.A. Butterfield, Spin labeling in disease, Biol. Mag. Reson. 4 (1982) 1. [31] D.A. Butterfield, Principles of biological membrane structure: alteration in the physical state of one side of the membrane by modulation of the physical state of the opposite side of the membrane, J. Membr. Sci. 53 (1990) 3. [32] D.A. Butterfield, J. Lee, Active site structure and stability of the thiol protease, papain, studied by electron paramagnetic resonance employing a methanethiosulfonate spin label, Arch. Biochem. Biophys. 310 (1994) 167. [33] D.S. Clark, J.E. Bailey, Characterization of hetrogeneous immobilized enzyme subpopulations using EPR spectroscopy, Biotech. Bioeng. 26 (1984) 231.
37
[34] Y. Song, G.E. Means, X. Wan, L.J. Berliner, A spin label study of immobilized enzyme spectral subpopulations, Biotech. Bioeng. 40 (1992) 306. [35] L.J. Berliner, Spin labeling in enzymology: spin-labeled enzymes and proteins, Meth. Enzymol 49 (1978) 418. [36] S.F. Oppenheim, G.P. Buettner, V.G.J. Rogers, Relationship of rotational correlation time from EPR spectroscopy and protein-membrane interaction, J. Membr. Sci. 118 (1996) 133. [37] R. Subramaniam, D.A. Butterfield, Structure-activity correlation of subtilisin immobilized on modified poly (ether) sulfone membrane by covalent and non-covalent linkages, in: D.A. Butterfield (Ed.), Biofunctional Membranes, Plenum Press, New York, 1996, p. 201. [38] D. Bhattacharyya, J.A. Hestekin, P. Brushaber, L. Collen, L.G. Bachas, S.K. Sikdar, Novel poly-glutamic acid functionalized microfiltration for sorption of heavy metals at high capacity, J. Membr. Sci. 141 (1998) 121. [39] D.A. Butterfield (Ed.), Biological and Synthetic Membranes, Wiley/Liss, New York, 1989. [40] R.F. Taylor (Ed.), Protein Immobilization: Fundamentals and Applications, Marcel Dekker, New York, 1991. [41] V. Schauer-Vukasinovic, S. Daunert, Affinity chromatography for recombinant peptides/proteins based on protein conformation changes, Biotechnol. Prog. 15 (1999) 513.
