Immobilization and Stabilization of Recombinant Multimeric Uridine

Sep 8, 2004 - Microbiologia, Via Ferrata 1, Universita` degli Studi, I-27100 Pavia, Italy, and ... practical challenge using strategies reported in th...
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Biomacromolecules 2004, 5, 2195-2200

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Immobilization and Stabilization of Recombinant Multimeric Uridine and Purine Nucleoside Phosphorylases from Bacillus subtilis Silvia Rocchietti,† Daniela Ubiali,‡ Marco Terreni,‡ Alessandra M. Albertini,§ Roberto Ferna´ ndez-Lafuente,| Jose´ M. Guisa´ n,| and Massimo Pregnolato*,‡ Innovate Biotechnology srl, Parco Scientifico Tecnologico, Strada Savonesa 9, I-15050 Rivalta Scrivia (AL), Italy, Dipartimento di Chimica Farmaceutica, Pharmaceutical Biocatalysis Laboratories, Via Taramelli 12, Universita` degli Studi, I-27100 Pavia, Italy, Dipartimento di Genetica e Microbiologia, Via Ferrata 1, Universita` degli Studi, I-27100 Pavia, Italy, and Departamento de Biocata´ lisis, Instituto de Cata´ lisis Campus UAM, Cantoblanco, E-28049 Madrid, Spain Received April 20, 2004; Revised Manuscript Received July 16, 2004

We selected the PnpI/PupG (PNP) with specificity for ribo- and deoxyriboguanosine and ribo- and deoxyriboinosine and the Up/Pdp (UP) with specificity for uridine, thymidine, and deoxyuridine from the purine and pyrimidine salvage pathway of the Gram-positive bacterium Bacillus subtilis. Then, an extensive study of the UP (uridine phosphorylase) and PNP (purine nucleoside phosphorylase) immobilization and stabilization was carried out: optimal UP preparation was achieved by immobilization onto Sepabeads coated with poly(ethyleneimine) and finally cross-linked with aldehyde dextran (UP-Sep-PEI-Dx); optimal immobilized PNP was prepared onto glyoxyl-agarose. Both derivatives were highly stable and active even under drastic experimental conditions (pH 10, 45 °C) unlike the free enzymes which were promptly inactivated. The derivatives prepared were successfully used in the synthesis of 2′-deoxyguanosine by enzymatic transglycosylation in aqueous solution between 2′-deoxyuridine and guanine. Introduction Modified nucleosides and nucleotides represent an important class of therapeutic agents with antiviral and antitumor activity; particularly, 2′-deoxynucleosides are key raw materials for the preparation of antisense drugs whose demand is rapidly increasing due to the recent success with their clinical development.1 The above-mentioned nucleosides can be prepared by chemical synthesis, generally by a glycosylation reaction;2 however, this approach requires timeconsuming multistage processes plagued by low yields and the formation of undesired byproducts. Particularly, the synthesis of purine 2′-deoxynucleosides still remains a practical challenge using strategies reported in the literature, since the reaction of purine base and activated 2′-deoxyribose produces both anomeric isomers and regioisomers.3 Alternatively, 2′-deoxynucleosides can be prepared via an enzymatic process, that is, by a transglycosylation reaction between a nucleoside and a purine or pyrimidine base in aqueous solution catalyzed by nucleoside phosphorylases or microorganisms that produce them.4 The use of biological catalysts may offer considerable advantages over chemical synthesis, such as stereo- and regioselectivity, greater reaction rate, and reduction of the preparation and purification steps. Specifically, the coupling * Corresponding author. Phone: +39-0382-507583. Fax: +39-0382507889. E-mail: [email protected]. † Innovate Biotechnology srl. ‡ Pharmaceutical Biocatalysis Laboratories. § Universita ` degli Studi. | Instituto de Cata ´ lisis Campus UAM.

of uridine phosphorylase (UP, EC 2.4.2.3) and purine nucleoside phosphorylase (PNP, EC 2.4.2.1) efficiently transfers a sugar moiety from a donor nucleoside to an acceptor base and has been extensively described.5-10 The enzymatic transglycosylation reactions can be performed by using bacterial cell pastes in which the required enzymatic activity has been induced, by purified free enzymes or, possibly, immobilized. Bacterial cell or extracted and purified phosphorylases are conveniently employed to carry out transglycosylation reactions at laboratory scale.11 The use of immobilized enzymes may be advantageous. Properly designed immobilization of enzymes on solid supports enables the reuse of the catalyst and may increase the stability of many enzymes under a wide range of experimental conditions.12 Stability limitations that characterize the enzymes may play a pivotal role when drastic reaction conditions are required for the solubilization of certain substrates and products at the high concentrations necessary to develop preparative or industrial processes. For instance, transglycosylation using guanine requires the use of alkaline pH and/or high temperature in order to solubilize suitable concentrations of this substrate and the products. Few examples describing the immobilization of UP and PNP on solid support are reported. It has been described that UP and PNP from Bacillus stearothermophilus have been immobilized on an anionic exchange resin (DEAE-Toyopearl) and used for the synthesis of 5-methyluridine starting from inosine as sugar donor.13 However, even if the optimal temperature of the immobilized enzymes was shifted to a higher temperature (probably due to a stabilization induced

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by the immobilization), the optimal pH was very narrow in comparison with the crude enzymes. More recently, Zuffi et al.14 described the coimmobilization of crude preparations from Escherichia coli containing UP and PNP on epoxyactivated solid supports by covalent linkages. However, the effect of the immobilization on the enzyme stability was not assessed in this interesting paper, because its goal was to use the enzymes at pH 7, where the enzyme stability was very high. In this work, we have studied the multimeric enzymes PnpI/PupG (purine nucleoside phosphorylase with specificity for ribo- and deoxyriboguanosine and ribo- and deoxyriboinosine) and Up/Pdp (pyrimidine nucleoside phosphorylase with specificity for uridine, thymidine, and deoxyuridine) from the purine and pyrimidine salvage pathway of the Gram-positive bacterium Bacillus subtilis.15 Due to their multimeric nature,16 we studied a suitable approach to efficiently immobilize and stabilize these enzymes in order to have biocatalysts stable under the reaction conditions required to ensure the solubility of guanine. The stabilization of the quaternary structure of multimeric enzymes, which requires the correct assembly of the all subunits to preserve their activity, is a complex goal.17-19 The potential dissociation into the single constitutive monomers causes the loss of activity. This dissociative process is usually favored by extreme pH values, high temperature, and the presence of organic solvents, conditions often necessary to ensure the solubility of poorly water soluble substrates. Consequently, when working with multimeric enzymes, the immobilization technique must be able to bind all the subunits in order to keep the enzyme quaternary structure unmodified under the selected experimental conditions. To stabilize the multimeric UP and PNP from B. subtilis, we have used different immobilization strategies. The simplest one was ionic adsorption. However, the use of this technique with a support bearing the ionic groups directly bonded to the surface may establish a weak interaction with the enzyme. This drawback, which can restrict the experimental applicable conditions, may be settled by using supports coated with polycationic polymers, e.g., poly(ethylenimine), allowing the enzyme to be fully covered by the polymer.20,21 Further cross-linking with aldehyde dextran may permit the complete stabilization of any multimeric enzyme and fully prevent the enzyme desorption under any reaction conditions. We have also tried to get covalent multi-subunit attachment using two different supports: Eupergit C and Sepabeads (epoxy hydrophobic resins), and agarose beads activated with glyoxyl groups (glyoxyl-agarose), a hydrophilic matrix. The mechanism of immobilization on both supports is very different. On epoxy resins, the immobilization occurs at first by hydrophobic interaction and, afterwords, by covalent-bond formation between the nucleophile groups of the enzyme and the epoxy functions of the support.22-26 Immobilization on glyoxyl-agarose beads occurs at alkaline pH, via the area richest in lysines on the proteins surface.27-29 A new approach has been developed for the stabilization of the multimeric structure of UP that was successfully used to improve the biosynthetic process for the preparation of

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2′-deoxynucleosides. Particularly, we investigated the synthesis of 2′-deoxyguanosine (2′-dG) starting from 2′-deoxyuridine (2′-dU) without isolation of the intermediate 2′deoxy-D-ribose-1-phosphate (one-pot synthesis). The UP and PNP derivatives, prepared according to the most appropriate immobilization technique, were able to catalyze this transglycosylation even at high temperature (45 °C) and strongly alkaline pH (pH 10), conditions required to operate in the presence of a high concentration of guanine (G), almost water insoluble under mild conditions (room temperature and pH 7). The results were compared with those obtained using the free enzymes or immobilized by ionic adsorption on DEAE. Experimental Section Materials and Methods. Ionic agarose (DEAE Sepharose CL-4B) was from Bio-Rad (Madrid, Spain); Eupergit C was from Rohmpharma Rohm GmbH (Darmstadt, Germany); agarose (Sepharose CL-4B) was purchased from Amersham Pharmacia Biotech (Uppsala, Sweden); Sepabeads FP-EC3 was kindly provided by Resindion Mitsubishi Chemical Co. (Binasco, Milano, Italy). Nucleosides, uracil, guanine, and hypoxanthine were supplied by Pro.Bio.Sint. (Varese, Italy). All reagents and solvents were of commercial quality and were not further purified before use. Activity assays were performed on a Shimadzu spectrophotometer UV 1601 (UVProbe 1.0 Shimadzu); reactions were monitored by HPLC (Multi HSM Manager Merck Hitachi D-7000). Cloning, expression, and purification of UP and PNP from B. subtilis15 were carried out as previously described.30 All experiments were highly reproducible, with an experimental error under 5%. Immobilization of UP and PNP on Eupergit C or Sepabead FP-EC3. Epoxy resin (Eupergit C or Sepabead FP-EC3, 1 g) was suspended in 2 M potassium phosphate buffer (10 mL) at pH 7.50 and room temperature. A suitable amount of UP or PNP solution (20 units) was added and the immobilization mixture was kept under mechanical stirring for 24 h. Then the derivative was filtered and washed with deionized water. Immobilization of UP on DEAE-Sepharose CL-4B. DEAE-Sepharose CL-4B (1 g) was suspended in 5 mM potassium phosphate buffer (10 mL) at pH 7.50 and room temperature. Then, a suitable amount of UP solution (20 units) was added. After 1 h under mechanical stirring the derivative was filtered and washed with deionized water. Immobilization of UP on Glyoxyl-agarose Gel. Glyoxylagarose gel (1.4 mL), prepared as previously described,26 was suspended in 50 mM potassium phosphate buffer at pH 10.05. After the addition of a suitable amount of UP solution (20 units), the suspension (14 mL; Vgel/Vtot ) 1/10) was kept under mechanical stirring at room temperature for 2 h. Then, 14 mg of NaBH4 (1 mg/mL suspension) was added and the reduction was carried out for 30 min. The derivative was finally filtered and washed with 10 mM potassium phosphate buffer at pH 5 and then with deionized water. Immobilization of UP on Sepabead-PEI (Sep-PEI). The activated support Sep-PEI was prepared as described below. Sepabead FP-EC3 (25 g) was added to a 10% solution of poly(ethyleneimine) (MW ) 20 000) in 1 M NaCl (pH 11).

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UP and PNP Immobilization and Stabilization

The suspension was kept under mechanical stirring for 24 h and then the support was filtered and washed with the solution of NaCl and with deionized water. The activated support (1 g) was suspended in 5 mM potassium phosphate buffer at pH 7.5 with a suitable amount of UP solution (20 units; total volume ) 14 mL; Vgel/Vtot ) 1/10) and kept under mechanical stirring for 30 min. The derivative was filtered and washed with deionized water. Stabilization of UP Sep-PEI Derivative with Dextran (Sep-PEI-Dx). Dextran (1.67 g, MW ) 19 500) was suspended in 50 mL of deionized water and 0.872 g of sodium periodate was added to obtain the partially oxidized (20%) dextran. The reaction was carried out for 2 h at room temperature and immediately dialyzed (3500 Da MWCO) with deionized water. Dextran-dialyzed solution (1.4 mL) was added to 14 mL of immobilization suspension of SepPEI prepared as reported above. After 1 h, 15.4 mg of NaBH4 was added (1 mg/mL suspension) and the reduction was carried out for 30 min. The derivative was filtered and washed with 10 mM potassium phosphate buffer (pH 5) and then with deionized water. Immobilization of PNP on Glyoxyl-agarose Gel. Glyoxyl-agarose beads (1.4 mL), prepared as previously described,26 were suspended in 50 mM potassium phosphate buffer (pH 10.05). After the addition of 100 mM MgCl2 (140 µL), inosine (38 mg), and Triton X-100 5% solution (1.4 mL), a suitable amount of PNP solution was added (20 units). The suspension (14 mL; Vgel/Vtot ) 1/10) was kept under mechanical stirring at room temperature for 2 h. Then, 14 mg of NaBH4 (1 mg/mL suspension) was added and the reduction was carried out for 30 min. The derivative was finally filtered, washed with 10 mM potassium phosphate buffer (pH 5), and then with deionized water. Activity Assay of PNP. The activity assay was carried out optimizing the procedure previously reported.31 In a 1.5 mL quartz cuvette with a 1-cm light path, 0.5 M potassium phosphate buffer at pH 7.0 (400 µL), 20 mM inosine (100 µL), xantine oxidase 0.2 U/mL (100 µL), and 300 µL of deionized water was mixed to allow a final volume of 0.9 mL. The reaction started by addition of a solution of PNP (100 µL). The activity was calculated from the increase in absorbancy at 300 nm ( ) 8828). Activity Assay of UP. The activity assay was carried out by optimizing the procedure previously reported.32 In a final volume of 830 µL, 0.5 M potassium phosphate buffer at pH 7.4 (230 µL), 100 mM 2′-deoxyuridine (50 µL), and deionized water (550 µL) were mixed. After the addition of a solution of UP (100 µL), the mixture was incubated at 37 °C for 15 min. The reaction was stopped by addition of 10 M NaOH (70 µL). The test solution was read against its control at 297 nm in cuvette of 1-cm light path ( ) 1912). General Procedure for the Enzymatic Synthesis of 2′Deoxyguanosine (2′-dG). A solution of 10 mM potassium carbonate buffer at pH 10 (25 mL) containing 570.5 mg of 2′-deoxyuridine, 0.375 mL of guanine solution (50 mM at pH 11) in the same buffer, and 68 mg of potassium phosphate was kept at 45 °C under mechanical stirring. The final pH was set up to 10 with dilute NaOH. The Sep-PEI-Dx derivative of UP (14 units) and PNP immobilized on glyoxyl-

Table 1. Immobilization-Stabilization of UP (Entries 1-6) and PNP (Entries 7-11) at a Theoretical Load of 20 U/mL or U/g entry 1 2 3 4 5 6 7 8 9

support Eupergit C glyoxyl-agarose DEAE-agarose Sepabeads Sep-PEI Sep-PEI-Dx Eupergit C glyoxyl-agarose glyoxyl-agarosec

additive

% immobil 100 100 100 96 100 100 100 0 50

activitya 2.4 0 20 0 20 14 1.6 0 10

% yieldb 12 0 100 0 100 78 8 0 50

MgCl2, inosine MgCl2, inosine, Triton X-100 d 10 glyoxyl-agarose MgCl2, inosine, 66 7.2 36 Triton X-100 e 11 glyoxyl-agarose MgCl2, inosine, 76 4 20 Triton X-100 a The activity of the enzyme derivative was expressed as U/mL or U/g for agarose or epoxy derivatives, respectively. b Evaluated as expressed activity. c Immobilization time 0.5 h. d Immobilization time 2 h. e Immobilization time 5 h.

agarose gel (30 units) was added to the suspension. The remaining solution of guanine (24.625 mL) was automatically added dropwise to the reaction mixture over 8 h (velocity 0.02-0.04 mL/min). The pH was kept at 10 with 2 N HCl by automated titration. The reaction was monitored by HPLC. The column was a RP select B Lichrocart 60 C8 (Merck, Darmstadt, Germany): eluent, 0.02 M KH2PO4 buffer/90% MeOH (97:3); flow, 1 mL/min; λ, 260 nm; T ) 35 °C. The suspension was stirred for 24 h and then stopped by filtration of the enzymatic derivatives under reduced pressure. After washing the derivatives with 10 mM potassium carbonate buffer (pH 10), the solution was cooled to 4 °C and slowly acidified to pH 5.5 until the formation of a white precipitate. The solid was filtered and characterized by HPLC as 2′deoxyguanosine (elution time 12.06 min, purity 97%), yield ) 92%. Results and Discussion Enzymes. PNP (PnpI/PupG) is one of the two purine nucleoside phosphorylases of B. subtilis that specifically catalyzes the cleavage of the glycosidic bond of inosine/ guanosine and 2′-dI/2′-dG in the presence of inorganic orthophosphate to generate the purine base and ribose or deoxyribose-1-phosphate. UP (Up/Pdp) is the only pyrimidine nucleoside phosphorylase of B. subtilis that uses uridine, thymidine, and deoxyuridine as pyrimidine source in the salvage pathway33 catalyzing the phosphorolytic cleavage of pyrimidine nucleoside to the pyrimidine base and deoxyribose- or ribose-1-phosphate. Both the enzymes were expressed from genes cloned in E. coli, purified, and characterized.34 The subunit composition of the two enzymes was confirmed both by aldehyde-dextran cross-linking,35 followed by SDS-PAGE of the complex, and by gel filtration, showing that PNP was a tetramer according to Bzowska et al.16 and suggesting a trimeric organization for UP. Immobilization-Stabilization of UP. For UP immobilization, commercially available supports such as ionic agarose (DEAE Sepharose CL-4B), epoxy resins (Eupergit C or Sepabeads FP-EC3), or agarose gel (Sepharose 6B CL) were used in order to study the effect of different matrixes and immobilization mechanisms. As reported in Table 1 (entries 1-6), the best result, as expressed activity, was obtained by ionic adsorption on

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Figure 1. Stability of UP: pH 10 and 45 °C.

DEAE. However, this derivative was particularly unstable (Figure 1) under extreme reaction conditions (pH 10 and 45 °C). The loss of activity was very similar to that observed for the free enzyme, and a complete inactivation occurred after 2 h. Also the immobilization on Eupergit C or Sepabeads afforded the complete immobilization of the protein, but the expressed activity was very poor (12% for Eupergit C) or negligible (Sepabeads). The very poor activity deriving from covalent immobilization may be ascribed to the distortion of the protein structure caused by the reaction between the enzyme and the support. Eupergit C derivative was also tested at 45 °C and pH 10 (Figure 1), resulting in poor stability. Using glyoxyl-agarose,

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the resultant derivative was completely inactive due to the reduction reaction with sodium borohydride necessary to obtain the formation of covalent bonds. To optimize the ionic adsorption of the enzyme, Sepabeads was derivatized with poly(ethyleneimine) (PEI; MW ) 20000), affording a support surface coated with a flexible cationic polymer (Figure 2, Sep-PEI) able to establish a nondistorting and very strong ionic interaction with the protein, according to a strategy previously reported for other enzymes.21 With this support, a complete immobilization of UP was achieved without any loss of activity (immobilization yield 100%). The stability of the Sep-PEI derivative at high pH and temperature was much greater than that observed with the DEAE derivative or the free enzyme (Figure 1). To further increase the stability of the Sep-PEI derivative, it was treated with polyaldehyde macromolecules (20% oxidized dextran) that, reacting both with the free amino groups of the enzyme and PEI, afforded a covalent multipoint cross-linking between the protein subunits and the support (Figure 2). The activity of this enzyme derivative (Sep-PEI-Dx) was very high (78%) and the stability increased (Figure 1) compared with the non-cross-linked derivative (Sep-PEI).

Figure 2. Stabilization of multimeric enzymes: immobilization onto Sepabeads derivatized with PEI and stabilized with dextran.

UP and PNP Immobilization and Stabilization

Figure 3. Stability of PNP: pH 10 and 45 °C.

The stabilized UP derivative maintained almost the total activity for 6 h at pH 10 and 45 °C (Figure 1). This treatment seemed to stabilize the multimeric structure of the enzyme: no enzyme subunit was detected in the supernatant of 9 M guanine boiled derivatives.21 Immobilization-Stabilization of PNP. As reported in Table 1 (entries 7-11), also for PNP immobilization the complete immobilization of the protein was obtained on the epoxy resin Eupergit C, but the expressed activity was very poor (8%) as well as its stability (Figure 3). When using glyoxyl-agarose, the addition of MgCl2 (1 mM) and inosine (10 mM) was necessary to stabilize the enzyme during the immobilization process performed at pH 10 and low ionic strength. After 24 h, the enzyme was not immobilized. When a surface-active agent, such as Triton X-100, was added to the immobilization solution, the percentage of immobilized enzyme increased. Under these conditions, 50% of the enzyme was immobilized after only 30 min without loss of activity (50% yield). This suggests that an aggregation of the proteins may occur in absence of detergent and the aggregates cannot diffuse inside of the porous structure of the support. As the immobilization time proceeded, the percentage of immobilization increased (76% after 5 h), but a progressive decrease in the expressed activity was observed (20% yield), probably because of the distortion of the protein due to its reaction with the support. Considering the stability (Figure 3), it appeared that the longer the immobilization time, the greater the derivative stability (pH 10 and 45 °C). A compromise between the expressed activity and the stability was found after 2 h of immobilization, which led to 36% expression of the initial activity. This derivative kept 44% of its activity after 24 h at pH 10 and 45 °C, whereas the free PNP was completely inactivated in a few minutes (Figure 3). One-Pot Synthesis of 2′-dG. The optimal biocatalysts were used for the one-pot synthesis of 2′-dG in fully aqueous medium with 2′-dU as donor of the carbohydrate moiety and guanine (G) as acceptor. However, the enzymatic transglycosylation with guanine as purine base was an obstacle for application to scalable preparations due to its very poor water solubility under mild reaction conditions (room temperature and neutral pH) suitable for working with nonstabilized enzyme preparations. The solubility of G is favorably affected by high pH and high temperature; to ensure a prolonged stability of the enzymatic derivatives, the optimal settlement was to perform the reaction at pH 10 and 45 °C. When the reaction was

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completed (90% conversion), the enzyme derivatives were removed by filtration and the desired product 2′-dG was easily isolated by precipitation from the solution containing uracil and residual 2′-dU and G: 2′-deoxyguanosine was precipitated from the reaction mixture as a white solid at pH ) 5.5 and T ) 4 °C (97% purity HPLC). The enzymatic transglycosylation catalyzed by the free enzymes was not successful because of the immediate inactivation of the non-immobilized catalysts, in agreement with the stability data (Figures 1 and 3). In conclusion, the stabilization of the two multimeric enzymes considered in this work allowed us to perform the synthesis of 2′-dG in high yield and concentration, otherwise impossible to be obtained by free or nonstabilized enzyme preparations. In particular, a new approach has been developed for the stabilization of the multimeric structure of UP based on the adsorption of the protein on a polymeric bead followed by the chemical cross-linking with tailor-made aldehyde-dextran. Acknowledgment. We thank Pro.Bio.Sint. (Euticals Group), Varese, Italy, for financial support. References and Notes (1) Komatsu, H.; Awano, H.; Tanikawa, H.; Itou, K.; Ikeda, I. Nucleosides, Nucleotides Nucleic Acids 2001, 20 (4-7), 1291-1293. (2) (a) Hilbert, G. E.; Johnson, T. B. J. Am. Chem. Soc. 1930, 52, 44894494. (b) Niedballa, U.; Vorbruggen, H. J. Org. Chem. 1974, 39, 3654-3674. (c) Jungmann, O.; Pfleiderer, W. Tetrahedron Lett. 1984, 25, 5017-5018. (3) Kawakami, H.; Matsushita, H.; Naoi, Y.; Itoh, K.; Yoshikoshi, H. Chem. Lett. 1989, 1, 235-238. (4) Krenitsky, T. A.; Koszalka, G. W.; Tuttle, J. V. Biochemistry 1981, 20 (12), 3615-3621. (5) Hennen, W. J.; Wong, C.-H. J. Org. Chem. 1989, 54, 4692-4695. (6) Cotticelli, G.; Magrı`, P.; Grisa, M.; Orsini, G.; Tonon, G.; Zuffi, G. Nucleosides Nucleotides 1999, 18 (4-5), 1135-1136. (7) Bestetti, G.; Calı`, S.; Ghisotti, D.; Orsini, G.; Tonon, G.; Zuffi G. WO-2000039307-A2, 2000. (8) Takashi, T.; Kanisuke, I.; Yokozeki, K. EP/916735A1, 1999. (9) Kojima, E.; Yoshioka, H.; Fukinbara, H.; Murakami, K. US-5563049, 1996. (10) Yokozeki, K.; Shirae, H.; Shiragami, H.; Irie, Y.; Yasuda, N.; Otani, M.; Tanabe, T. US Patent 4,835,104, 1989. (11) Hutchinson, D. W. Trends Biotechnol. 1990, 8 (12), 348-353. (12) (a) Gupta, M. N. Biotechnol. Appl. Biochem. 1991, 14 (1), 1-11. (b) Klibanov, A. M. AdV. Appl. Microb. 1983, 29, 1-28. (c) Gianfreda, L.; Scarfi, M. R. Mol. Cell. Biochem. 1991, 100 (2), 97128. (13) Hori, N.; Watanabe, M.; Sunagawa, K.; Uehara, K.; Mikami, Y. J. Biotech. 1991, (17), 121-131. (14) Zuffi, G.; Ghisotti, D.; Oliva, I.; Capra, E.; Frascotti, G.; Tonon, G.; Orsini, G. Biocatal. Biotransf. 2004, 22 (1), 25-33. (15) Switzer, R. L.; Zalkin, H.; Saxild, H. H. In Bacillus subtilis and Its Closest RelatiVes: from genes to cells; Sonenshein, A. L., et al., Eds.; ASM Press: Washington, DC, 2002; pp 255-269. (16) Bzowska, A.; Kulikowska, E.; Shugar, D. Pharmacol. Ther. 2000, 88, 349-425. (17) Ferna´ndez-Lafuente, R.; Rodrı`guez, V.; Mateo, C.; Penzol, G.; Herna´ndez-Justiz, O.; Irazoqui, G.; Villarino, A.; Ovsejevi, K.; Batista, F.; Guisa´n, J. M. J. Mol. Catal. B: Enzymol. 1999, 7, 181189. (18) Balca˜o, V. M.; Mateo, C.; Ferna´ndez-Lafuente, R.; Malcata, F. X.; Guisa´n, J. M. Biotechnol. Prog. 2001, 17 (3), 537-542. (19) Ferna´ndez-Lafuente, R.; Herna´ndez-Ju´stiz, O.; Mateo, C.; Ferna´ndezLorente, G.; Terreni, M.; Alonso, J.; Garcia-Lo´pez, J. L.; Moreno, M. A.; Guisa´n, J. M. Biomacromolecules 2001, 2, 95-104. (20) Torres, R.; Mateo, C.; Fuentes, M.; Palomo, J. M.; Ortiz, C.; Ferna´ndez-Lafuente, R.; Guisa´n, J. M.; Tam, A.; Daminati, M. Biotechnol. Prog. 2002, 18 (6), 1221-1226. (21) Mateo, C.; Abian, O.; Ferna´ndez-Lafuente, R.; Guisa´n, J. M. Biotechnol. Bioeng. 2000, 68, 98-105.

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