Design of a Specific Peptide Tag that Affords Covalent and Site

Site-Specific Enzyme Immobilization Catalyzed by Microbial ... Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 6-1...
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Biomacromolecules 2005, 6, 2299-2304

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Design of a Specific Peptide Tag that Affords Covalent and Site-Specific Enzyme Immobilization Catalyzed by Microbial Transglutaminase Jo Tominaga,† Noriho Kamiya,*,† Satoshi Doi,† Hirofumi Ichinose,‡ Tatsuo Maruyama,† and Masahiro Goto*,†,‡ Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan, and PRESTO, JST, 4-1-8 Honmachi, Kawaguchi, Saitama 332-0012, Japan Received March 12, 2005; Revised Manuscript Received May 13, 2005

Transglutaminase-mediated site-specific and covalent immobilization of an enzyme to chemically modified agarose was explored. Using Escherichia coli alkaline phosphatase (AP) as a model, two designed specific peptide tags containing a reactive lysine (Lys) residue with different length Gly-Ser linkers for microbial transglutaminase (MTG) were genetically attached to N- or C-termini. For solid support, agarose gel beads were chemically modified with β-casein to display reactive glutamine (Gln) residues on the support surface. Recombinant APs were enzymatically and covalently immobilized to casein-grafted agarose beads. Immobilization by MTG markedly depended on either the position or the length of the peptide tags incorporated to AP, suggesting steric constraint upon enzymatic immobilization. Enzymatically immobilized AP showed comparable catalytic turnover (kcat) to the soluble counterpart and comparable operational stability with chemically immobilized AP. These results indicate that attachment of a suitable specific peptide tag to the right position of a target protein is crucial for MTG-mediated formulation of highly active immobilized proteins. Introduction Proteins are versatile functional biomolecules and have a wide range of applications. Immobilization of proteins onto solid supports is a promising biotechnological application because it is easy to separate proteins from the reaction media, and repeated use of the protein function of interest is possible. In particular, immobilized enzymes have already been extensively investigated.1,2 The covalent attachment of enzymes to solid supports is attractive from industrial viewpoints because stable linkage formation can diminish the leakage of enzymes. To form a covalent bond between enzymes and solid supports, a number of techniques has been proposed for the chemical derivatization of the support surface.3-5 Although the feasibility of chemical immobilization has attracted much attention, it also often results in the inactivation of enzymes to some extent because the chemical reaction between enzymes and the activated support surface can be difficult to control. This, in turn, leads to the random orientation of immobilized enzymes. To alleviate inactivation during the immobilization process, several approaches have recently been proposed for enzyme immobilization. For noncovalent and site-specific immobilization, monoclonal antibodies, which bind to an enzyme with high affinity without affecting its catalytic activity, were employed. Full catalytic activity and improved stability of a * To whom correspondence should be addressed. E-mail. (N.K.) [email protected] or (M.G.) mgototcm@ mbox.nc.kyushu-u.ac.jp. † Kyushu University. ‡ PRESTO.

target enzyme were reported when carriers were suitably formulated with the monoclonal antibodies.6 Affinity peptide tags, such as a polyhistidine tag (His-tag) or an Asp-TryLys-Asp-Asp-Asp-Asp-Lys (FLAG) were also used and were genetically attached to enzymes. In the former strategy, Histagged recombinant proteins were immobilized site-specifically to nickel-titanium dioxide film.7 In the latter case, the recombinant protein-FLAG conjugate was immobilized site-specifically using anti-FLAG antibody.8,9 For covalent and site-specific immobilization, site-directed mutagenesis can be used to introduce a cysteine (Cys) residue at a suitable position on proteins, and the resultant recombinant proteins can be site-specifically bound to supports displaying maleimide groups through the sulfhydryl group originating from the Cys residue.10,11 However, this strategy is limited to just those proteins that either lack or contain only one reactive Cys residue in the target proteins. As for covalent immobilization of His-tagged proteins, Mateo et al. developed a novel heterofunctional chelate-epoxy support.12 It is of great interest that their approach can realize site-specificity in chemical immobilization, which is usually based on random protein modifications. Recently, enzymatic strategies for protein immobilization using microbial transglutaminase (MTG) from Streptomyces mobaraensis have attracted much attention. MTG is a unique enzyme that catalyzes protein cross-linking between the -amino group of lysine (Lys) residues and the γ-carboxyamide group of glutamine (Gln) residues in certain peptides or proteins.13-17 As MTG-mediated posttranslational modification of proteins proceeds under mild conditions with high

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specificity, one can imagine that the MTG-catalyzed reaction is applicable to immobilization of functional proteins. In their pioneering work, Kamata et al. found that enzymes could be stably immobilized onto ion exchangers after physical absorption and subsequent MTG-treatment.18 Kawakita et al. succeeded in extending this concept to immobilization of an enzyme onto anion-exchange hollow-fiber membranes with exceptionally high enzyme loading.19 However, with the requirement of physical adsorption of enzymes prior to MTG treatment, it seemed that some portion of the enzymes worked as an inactive proteinaceous binder in these systems. Although immobilization of enzymes in a cross-linked protein network formed by MTG was demonstrated, the mode of immobilization is basically the entrapment of native proteins.20 To maximize the potential utility of MTG in enzyme immobilizations, we have genetically incorporated a specific peptide tag to a target enzyme and attempted peptide tagdirected covalent protein immobilization by MTG. In previous studies, we employed chemically modified21 and physically coated22 solid supports with casein, a good MTG substrate, as a solid surface displaying reactive Gln for MTG. Escherichia coli alkaline phosphatase (AP) tagged with a specific peptide tag including reactive Lys for MTG was successfully immobilized to these supports.21,22 Our strategy is basically applicable to any recombinant proteins that afford the incorporation of specific peptide sequences available for MTG catalysis. However, quantitative evaluation of the enzymatic immobilization strategy as compared with conventional methods has not yet been undertaken. This study aimed to explore the factors affecting MTGmediated protein immobilization. Among inorganic and organic materials, we chose agarose, which is routinely used for the preparation of solid protein formulations, as an immobilization template.23,24 Agarose beads consist of a three-dimensional network of highly hydrophilic and extremely inert fibers, the surface of which is essentially covered by hydroxyl groups, which can be easily activated for covalent immobilization of proteins, thus providing a high specific area for the immobilization. In fact, chemically activated agarose beads (e.g., chemically activated Sepharose) are commercially available, and proteins are easily but randomly immobilized on the supports. In the present study, N-hydroxysuccineimide (NHS)-activated agarose beads were grafted with β-casein to display reactive Gln residues for MTG-mediated immobilization on the support surface. For an enzyme to be immobilized, a set of recombinant APs with a variety of peptide tags was prepared. Using these, we investigated how the position and linker length of a peptide tag incorporated to AP affected the immobilization process and the catalytic performance of the resultant immobilized formulations. The results obtained here indicate the importance of designing a suitable peptide tag to enhance the utility of MTG-mediated protein immobilization and its applications. Materials and Methods Materials. MTG was provided by Ajinomoto Co., Inc. (Japan), and its activity was measured by the colorimetric

Tominaga et al. Table 1. Recombinant APs Prepared in This Studya

a Amino acid sequences of N- or C-terminal regions are shown with one-letter codes for each type of AP prepared. The omitted region had the same sequence for all types of AP.

hydroxamate procedure with N-carbobenzyloxy-glutaminylglycine as previously described.25 One unit (U) of activity was defined as the amount of enzyme that catalyzes the formation of 1 µmol of hydroxamate per minute using L-glutamic acid γ-monohydroxamate as standard. NHSactivated agarose was purchased from Amersham Biosciences. Bovine β-casein was purchased from SigmaAldrich. All other reagents were of commercially available analytical grade. Preparation of Recombinant AP Tagged with Specific Peptide Linker for MTG. Two specific peptide tags, MKHKGS (abbreviated as K6-tag) or MKHK(GGGS)2GS (abbreviated as K14-tag), were genetically attached to the N- or C-terminus of AP. The resultant N-terminal K6-tagged, K14-tagged APs and C-terminal K6-tagged AP were designated as NK6-AP, NK14-AP, and CK6-AP, respectively (Table 1). NK6-AP was prepared as previously reported.21 The preparation of CK6-AP and NK14-AP was as follows. A DNA fragment encoding CK6-AP was amplified by the polymerase chain reaction (PCR) using pET22b-AP as the template DNA. The primer nucleotide sequences used for PCR were 5′-GGG GGG ATC CGC ACC ACC ACC ACC ACC ACA CCC CAG AAA TGC CT-3′ and 5′-CCC CCA AGC TTT CAA GAA CCT TTA TGT TTC ATT TTC AGC CCC AGA GC-3′. The resultant gene fragment encoding CK6-AP with BamHI and HindIII sites was cloned into a bacterial expression plasmid vector, pET22b(+) (Novagen), by digestion with the restriction enzymes (pET22b-CK6-AP). Next, a DNA fragment encoding NK6-AP was amplified by PCR using pET22b-NK6-AP as the template DNA. The primer nucleotide sequences used for the first PCR were 5′TAA TAC GAC TCA CTA TAG GG-3′ and 5′-GCT AGT TAT TGC TCA GCG-3′. The resultant gene fragment encoding NK6-AP was cloned into a plasmid vector, pUC18 (Novagen), by SmaI digestion (pUC18-NK6-AP). To attach the K14-tag with AP, inverse PCR was conducted using pUC18-NK6-AP as the template with the primers 5′-GGT GGT GGT TCC GGA TCC ACC CCA TCC ATG CC-3′ and 5′-GGA ACC ACC TCC TTT ATG TTT CAT GGC CAT-3′. The PCR product was self-ligated (pUC18-NK14AP). Next, a DNA fragment encoding NK14-AP was amplified by the PCR using pUC18-NK14-AP as the template DNA. The primer nucleotide sequences used for this PCR were 5′-CAG GAA ACA GCT ATG AC-3′ and 5′-GTA AAA CGA CGG CCA GT-3′. The resultant gene fragment encoding NK14-AP with the NdeI and XhoI sites was cloned into pET22b(+) digested with the same restriction enzymes (pET22b-NK14-AP). CK6-AP and NK14-AP

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Scheme 1. Schematic Illustration of the Preparation Procedures for Immobilized Recombinant APsa

a (a) Chemical immobilization was performed using NHS-activated agarose gel beads. (b) MTG-mediated enzymatic immobilization was performed using β-casein-grafted agarose gel beads prepared with the same NHS-activated template.

were expressed in E. coli strain BL21. The fusion proteins obtained were purified by the hexahistidine-tag (His-tag) attached to the N-terminus of CK6-AP and C-terminus of NK14-AP. The purified proteins were employed as CK6AP and NK14-AP, respectively. Measurement of AP Activity and Determination of Kinetic Parameters. The catalytic activity of AP was measured with p-nitrophenyl phosphate (p-NPP) as a substrate. To 1 mL of 1 M Tris-HCl buffer (pH 8.0), the hydrolysis of p-NPP (1 mM) was initiated by the addition of an AP formulation at 25 °C under vigorous stirring (1.4 µg/mL soluble recombinant AP; 10 µL/mL recombinant AP immobilized agarose). The initial activity was determined by detecting the increase in the absorbance at 410 nm (derived from p-nitrophenol (p-NP)) by a UV-vis spectrophotometer (Jasco V-570, Tokyo). One unit (U) of activity was defined as the amount of enzyme that catalyzes the formation of 1 µmol of p-NP per minute. Kinetic parameters were determined by changing the substrate concentration, ranging from 0.01 to 1 mM. Kinetic parameters were determined by fitting the experimental data obtained to the double reciprocal plot of the Michaelis-Menten equation (i.e., a Lineweaver-Burk plot). Preparation of Casein-Grafted Agarose Beads. β-Casein was chemically immobilized on NHS-activated agarose gel beads as outlined in Scheme 1. The immobilization was initiated by the addition of β-casein (10 mg/mL) dissolved in 1 mL of 100 mM phosphate buffer (pH 7.0) to 0.1 mL of NHS-activated agarose (NHS-activated Sepharose 4 Fast Flow, Amersham Biosciences). After incubation for 3 h at 25 °C, agarose beads were recovered by centrifugation (8000 rpm for 5 min) and added to 1 mL of 1 M ethanolamine to block the residual active NHS groups. After incubation for 3 h, agarose beads were again recovered by centrifugation and washed with 1 mL of 100 mM Tris-HCl buffer (pH 8.0) and 1 mL of 100 mM acetic acid buffer (pH 4.0). This cycle was repeated three times to get rid of any physically adsorbed residual β-casein. In each case, the concentration of β-casein in the supernatant was measured by bicinchonic acid protein assay (BCA assay kit, Sigma), and the amount of β-casein immobilized onto the supports was determined by comparing it with the initial β-casein concentration before the im-

mobilization procedures. The resultant solid formulations were employed as casein-grafted agarose beads for subsequent experiments. Immobilization of Recombinant APs to Casein-Grafted Agarose Beads by MTG. Chemically immobilized AP was prepared by the addition of recombinant AP (28 µg/mL) dissolved in 0.5 mL of 100 mM phosphate buffer (pH 7.0) to NHS-activated agarose (0.1 mL) at 25 °C (Scheme 1a). Chemically immobilized AP were recovered by centrifugation after incubation for 3 h and then washed with 1 mL of 1 M ethanolamine to block residual active NHS groups. After incubation for 3 h, the immobilized AP was recovered by centrifugation and washed with 1 mL of 100 mM Tris-HCl buffer (pH 8.0) and 1 mL of 100 mM acetic acid buffer (pH 4.0) three times, respectively. MTG-mediated immobilization of a recombinant AP to casein-grafted agarose beads (0.1 mL) was carried out by the addition of 0.5 mL of 100 mM Tris-HCl buffer (pH 8.0) containing a recombinant AP (28 µg/mL) and MTG (5.4 U/mL) at 25 °C (Scheme 1b). The immobilized AP was recovered by centrifugation after incubation for 3 h and washed by the same protocol with chemically immobilized APs. AP activity in the supernatant was measured to calculate the amount of recombinant APs immobilized onto the supports. Conjugation of Wild-Type and Recombinant APs to Soluble β-Casein by MTG. Wild-type, CK6-AP, NK6-AP, or NK14-AP (0.5 mg/mL) and β-casein (1 mg/mL) were dissolved in 100 mM Tris-HCl buffer at pH 8. The conjugation reaction was initiated by the addition of MTG (5.4 U/mL) at 25 °C. After incubation for 3 h, the reaction products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were stained with Coomassie Brilliant Blue (CBB), and the protein band intensity was quantitated by image analysis using software (CS analyzer, ATTO, Japan) on a personal computer. Continuous Use of Immobilized NK14-AP in a PackedBed Column Reactor. Continuous use of two immobilized NK14-AP formulations was investigated in a packed-bed column reactor. The column was packed with immobilized AP as prepared in Immobilization of Recombinant APs to Casein-Grafted Agarose Beads by MTG to a bed height of

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Table 2. Yields of Immobilized Wild-Type and Recombinant APs by Chemical or Enzymatic Manipulation protein

chemical immobilization (%)

MTG-mediated immobilization (%)

wild-type AP CK6-AP NK6-AP NK14-AP

92 96 94 93

9 12 36 71

2.5 cm (0.5 mL of NK14-AP immobilized agarose). The substrate solutions (10 mM) in 100 mM Tris-HCl buffer (pH 8.0) were fed continuously into the column using a peristaltic pump at the flow rate of 0.5 mL/min. Results and Discussion Design of Recombinant APs and the Solid Support Displaying Reactive Gln Residues. When considering the use of MTG in protein immobilization, the substrate accessibility of this enzyme26 needs to be taken into consideration to effectively immobilize target proteins. Therefore, we investigated how the position and length of Gly-Ser flexible peptide linkers affect MTG-mediated immobilization of alkaline phosphatase. For N-terminal peptide-tagged APs, peptide tags constituting six MKHKGS (K6-tag) and 14 MKHK(GGGS)2GS (K14-tag) amino acids were designed. The first Met residue is derived from the cleavage of the periplasmic signal peptide originating from the plasmid vector. The subsequent three amino acids were found to be reactive in MTG-mediated protein immobilization.21,22 To conduct site-specific immobilization of the recombinant APs through the specific peptide tags containing a reactive Lys residue, a solid support needs to display reactive Gln residues accessible to the active site of MTG for the crosslinking reaction. For this, β-casein was selected because it is a good MTG substrate27 and basically adopts a random coil conformation in solution.28 These characteristics of β-casein are considered to be suitable to display reactive Gln residues that are accessible for MTG on agarose beads. The chemical attachment of β-casein to NHS-activated agarose is directed to Lys residues of β-casein, so that Gln residues are left for the subsequent cross-linking of recombinant APs. Using the pair of recombinant APs and the casein-grafted agarose beads, we investigated the factors affecting MTGmediated protein immobilization. MTG-Mediated Enzyme Immobilization onto CaseinGrafted Agarose Beads. Table 2 shows the protein immobilization yields from chemical modification or MTGmediated modification. In the case of chemical immobilization, more than 90% of all wild-type and recombinant APs was immobilized, suggesting that chemical modification occurred equally toward Lys residues on the protein surface. By contrast, in MTG-mediated immobilization, a strong dependence of the peptide tags on the yield of the immobilization was evident. When comparing the short K6-tagged APs, NK6-AP showed a 3-fold higher immobilization yield than did CK6-AP, which showed a comparable yield of nonspecific adsorption of wild-type AP (Table 2), thus indicating that the position of the peptide tags is of great importance.

Figure 1. Comparison of specific activities of recombinant APs, among soluble (open bars), chemically immobilized (striped bars), and enzymatically immobilized (dark bars) formulations. Mean values and standard deviations (error bars) are depicted.

The results prompted us to test if the extension of a flexible linker inserted between the specific KHK sequence and the target protein would improve the immobilization yield. As a consequence, the highest immobilization yield was obtained (71%), indicating that the relaxation of steric hindrance around the reactive Lys residue can lead to efficient immobilization. It is worth noting that the order of reactivity of APs in conjugation with soluble β-casein, estimated by the image analysis (see Supporting Information), was of the same order as the immobilization efficiency: wild-type AP ∼ CK6-AP , NK6-AP (60% conversion) < NK14-AP (73% conversion). The comparable reactivity of recombinant APs both in homogeneous and solid-liquid heterogeneous systems suggests that enhancement of the intrinsic reactivity of substrate proteins is vital to achieve successful MTGmediated protein immobilization. However, for the reactivity of NK6- and NK14-APs, it was found that reactivity differences were more pronounced in the heterogeneous system (about 2-fold increase in yield, Table 2) than the homogeneous system (about 1.2-fold increase in yield), implying steric hindrance upon the immobilization of NK6AP. The immobilization yield with NK14-AP was lower yet than the case with chemical modification. Under the experimental conditions, an about 400-fold higher equivalent of β-casein than NK14-AP was present in the system. In terms of MTG-mediated immobilization, hydrolysis of reactive Gln to nonreactive Gln residues should be taken into consideration. The lower yield might be due to the lower accessibility of the macromolecular substrates (i.e., the recombinant APs) to the acyl-enzyme intermediate (i.e., β-casein-MTG complex) formed on the support surface, which can increase the competitive hydrolysis. Characterization of Immobilized AP Formulations. Figure 1 shows a comparison of the specific activities of soluble N-terminal peptide-tagged recombinant APs with the two immobilized forms. When chemically immobilized, the catalytic activities of both NK6- and NK14-APs were reduced to approximately 40% as compared with soluble

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Table 3. Kinetic Parameters of NK14-AP in Free and Chemically and Enzymatically Immobilized Formsa NK14-AP preparation

Km (µM)

kcat (s-1)

kcat/Km (M-1 s-1)

free chemical preparation enzymatic preparation

23 96 97

21 10 18

9.4 × 105 1.1 × 105 1.9 × 105

a The values with chemically and enzymatically immobilized forms are the apparent values obtained under the experimental conditions (see Materials and Methods for more detail).

forms. It appeared that random formation of covalent bonds between the surface amino groups of recombinant APs and NHS-esters on agarose beads could reduce the catalytic performance. On the other hand, in the case of MTGmediated immobilization, about 90% of the native activity was retained, suggesting the high catalytic performance of immobilized formulations. Since the active site of AP is located at the opposite side of its N-terminus,29 β-casein molecules attached through N-terminal K6- or K14-tags did not significantly affect the catalytic property of AP. It was found that the catalytic performance of immobilized formulations is independent of the length of flexible linkers, suggesting that the active site environment of AP in the two solid formulations is virtually identical. It is known that the homodimeric form is essential for the enzyme activity of AP,30 so the random, multipoint attachment of AP to the support may cause the disruption of the active homodimeric structure. By contrast, site-specific attachment of AP to grafted β-casein on agarose gel beads, which is considered to be quite flexible, could support the native dimeric form more easily than in the case of chemical immobilization. Kinetic Study of Immobilized NK14-AP Formulation. To gain further insight into factors affecting the catalytic performance of immobilized APs, kinetic analyses of soluble and two immobilized NK14-APs were conducted. The kinetic constants obtained are summarized in Table 3. It was found that apparent Km values of both chemically and enzymatically immobilized preparations were higher than the Km value of soluble NK14-AP. The increase in Km values was attributable to the lower accessibility of the substrate to the active site of the immobilized NK14-AP. On the other hand, the kcat value of immobilized NK14-AP prepared by MTG was higher than that of the chemically immobilized one and was about 86% of the soluble NK14-AP, in good agreement with the observation in Figure 1. The results clearly showed that MTG-mediated site-specific immobilization contributes to the retention of the basic catalytic process of AP. With respect to kcat/Km values, however, the catalytic efficiency of enzymatic preparations showed one-fifth of that of the aqueous preparations due to the increase of the Km value, whereas that of chemical preparations showed one-ninth. Kinetic analysis revealed that chemical immobilization negatively affects both Km and kcat values of NK14-AP, whereas MTG-mediated immobilization affords nativelike catalytic turnover of NK14-AP. Continuous Use of Immobilized NK14-AP. Proteins immobilized onto agarose beads are often used in a column reactor.31 To check the operational stability of immobilized APs, continuous use of the two covalently immobilized NK14-APs was investigated in a packed-bed column reactor.

Figure 2. Continuous use of immobilized NK14-APs prepared by chemical (open squares) and enzymatic (dark squares) modification.

As shown in Figure 2, both immobilized preparations showed no reduction in catalytic performance over a period of 20 days. It is worth noting that enzymatic preparation with the single-point attachment of NK14-AP per molecule ensures an operational stability of chemical preparation comparable with multipoint attachment to the supports.32 The results provide further evidence of the covalent bond formation of NK14-AP to the casein-grafted agarose beads and clearly show the potential of MTG-mediated enzyme immobilization. Conclusions In the present study, we demonstrated MTG-mediated sitespecific and covalent immobilization of recombinant AP to casein-grafted agarose beads. The immobilization yield of MTG-mediated immobilization was lower than that of chemical immobilization; however, the specific activity of immobilized recombinant APs approaches that of the soluble counterparts. Interestingly, the immobilization yield was strongly dependent on both the position and the linker length of the flexible specific peptide tag incorporated to the AP, and it appeared that longer flexible linkers are suitable for MTG-mediated immobilization. It was observed in this study that enzymatic immobilization especially benefits when target proteins are sensitive to chemical modification. The results obtained here clearly illustrate the advantages of MTGmediated immobilization of recombinant, multimeric proteins under mild conditions. Acknowledgment. We are grateful to Ajinomoto Co., Inc. for providing MTG samples. The present work was mainly supported by a Grant-in-Aid for Scientific Research (16760638) from the Ministry of Education, Culture, Science, Sports and Technology (MEXT) of Japan and by a grant from the Kyushu Industrial Technology Center (N.K.) and was partly supported by the 21st Century COE Program, Functional Innovation of Molecular Informatics from the MEXT of Japan (M.G.). Supporting Information Available. Figure showing SDS-PAGE analysis of MTG-mediated conjugation of three

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recombinant APs to β-casein in aqueous solution. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Chibata, I. Immobilized enzymes. Research and deVelopment; Kodansha: Tokyo, 1978. (2) Goldstein, L.; Katchalski-Katzir, E. In Immobilized enzyme principles. Series Applied Biochemistry and Bioengineering; Wingard, L. B., Jr., Katchalski-Katzir, E., Goldstein, L., Eds.; Academic Press: New York, 1976; Vol. 1, pp 1-22. (3) Tischer, W.; Kasche, V. Trends Biotechnol. 1999, 17, 326. (4) Katchalski-Katzir, E.; Kraemer, D. M. J. Mol. Catal., B 2000, 10, 157. (5) Mateo, C.; Torres, R.; Fernandez-Lorente, G.; Ortiz, C.; Fuentes, M.; Hidalgo, A.; Lopez-Gallego, F.; Abian, O.; Palomo, J. M.; Betancor, L.; Pessela, B. C. C.; Guisan, J. M.; Fernandez-Lafuente, R. Biomacromolecules 2003, 4, 772. (6) Solomon, B.; Pines, G.; Koppel, G.; Katchalski-Katzir, E. Biotechnol. Bioeng. 1986, 28, 1213. (7) Cass, A. E. G.; Zhang, J. K. Anal. Biochem. 2001, 292, 307. (8) Vishwanath, S. K.; Watson, C. R.; Huang, W.; Bachas, L. G.; Bhattacharyya, D. J. Chem. Technol. Biotechnol. 1997, 68, 294. (9) Wang, J.; Bhattacharyya, D.; Bachas, L. G. Biomacromolecules 2001, 2, 700. (10) Huang, W.; Wang, J.; Bhattacharyya, D.; Butterfield, L. G. Anal. Chem. 1997, 69, 4601. (11) Vishwanath, S.; Wang, J.; Bachas, D. A.; Butterfield, D. A.; Bhattacharyya, D. Biotechnol. Bioeng. 1998, 60, 608. (12) Mateo, C.; Fernandez-Lorente, G.; Cortes, E.; Garcia, J. L.; Fernandez-Lafuente, R.; Guisan, J. Biotechnol. Bioeng. 2001, 76, 269. (13) Kamiya, N.; Ogawa, T.; Nagamune, T. Biotechnol. Lett. 2001, 23, 1629. (14) Kamiya, N.; Tanaka, T.; Suzuki, T.; Takazawa, T.; Takeda, S.; Watanabe, K.; Nagamune, T. Bioconjugate Chem. 2003, 14, 351.

Tominaga et al. (15) Kamiya, N.; Takazawa, T.; Tanaka, T.; Ueda, H.; Nagamune, T. Enzyme Microb. Technol. 2003, 33, 492. (16) Takazawa, T.; Kamiya, N.; Ueda, H.; Nagamune, T. Biotechnol. Bioeng. 2004, 86, 399. (17) Tanaka, T.; Kamiya, N.; Nagamune, T. Bioconjugate Chem. 2004, 15, 491. (18) Kamata, Y.; Ishikawa, E.; Motoki, M. Biosci. Biotechnol. Biochem. 1992, 56, 1323. (19) Kawakita, H.; Sugita, K.; Saito, K.; Tamada, M.; Sugo, T.; Kawamoto, H. Biotechnol. Prog. 2002, 18, 465. (20) Josten, A.; Meusel, M.; Spener, F.; Haalck, L. J. Mol. Catal., B 1999, 7, 57. (21) Tominaga, J.; Kamiya, N.; Doi, S.; Ichinose, H.; Goto, M. Enzyme Microb. Technol. 2004, 35, 613. (22) Kamiya, N.; Doi, S.; Tominaga, J.; Ichinose, H.; Goto, M. Biomacromolecules 2005, 6, 35. (23) Barros, R. M.; Extremina, C. I.; Goncalves, I. C.; Braga, B. O.; Balcao, V. M.; Malcata, F. X. Enzyme Microb. Technol. 2003, 33, 908. (24) Tardioli, P. W.; Pedroche, J.; Giordano, R. L. C.; Fernandez-Lafuente, R.; Guisan, J. M. Biotechnol. Prog. 2003, 19, 352. (25) Folk, J. E.; Cole, P. W. J. Biol. Chem. 1965, 240, 2951. (26) Jong, G. A. H.; Wijngaards, G.; Boumas, H.; Koppelman, S. J.; Hessing, M. J. Agric. Food. Chem. 2001, 49, 3389. (27) O’Connell, J. E.; Kruif, C. G. Colloid Surf., A 2003, 216, 75. (28) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir 2000, 16, 2242. (29) Kim, E. E.; Wyckoff, W. J. Mol. Biol. 1991, 218, 449. (30) McComb, R. B.; Bowers, G. M.; Posen, S. Alkaline Phosphatase. Plenum Press: New York, 1979. (31) Kim, P.; Yoon, S. H.; Roh, H. J.; Choi, J. H. Biotechnol. Prog. 2001, 17, 208. (32) Suh, C. W.; Park, S. H.; Park, S. G.; Lee, E. K. Process. Biochem. 2005, 40, 1755.

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