Zirconium Phosphate and Modified Zirconium Phosphates as

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Langmuir 2002, 18, 8737-8742

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Zirconium Phosphate and Modified Zirconium Phosphates as Supports of Lipase. Preparation of the Composites and Activity of the Supported Enzyme Francesca Bellezza,† Antonio Cipiciani,*,† Umberto Costantino,*,‡ and M. Elena Negozio‡ Laboratorio di Chimica Organica and Laboratorio di Chimica Inorganica, Dipartimento di Chimica, Universita` di Perugia, Via Elce di Sotto, 06123 Perugia, Italy Received July 24, 2002. In Final Form: September 13, 2002 Biocomposites with enzymatic activity were obtained by adsorption of lipase from Candida rugosa on the surface of different layered zirconium phosphates and phosphonates such as R-zirconium hydrogenphosphate, solid dispersions of zirconium phosphate in silica, zirconium carboxyethanephosphonate, zirconium phosphate-carboxyethanephosphonate, zirconium benzenephosphonate, and zirconium phosphatebenzenephosphonate. All the supports were characterized for chemical composition, BET specific surface area, surface ion exchange capacity, and X-ray diffraction patterns. The adsorption process at 4 °C was studied as a function of time of equilibration of the support with the lipase solutions (0.5 mg/mL) and as a function of the amount of protein present in the equilibrating solution. The activities of biocomposites with the different supports, at different protein loadings, were obtained by determining the amount of acetic acid produced by catalyzed hydrolysis of p-nitrophenylacetate. The best results in terms of protein surface adsorption (29 mg of protein/g of support) and of catalytic efficiency (95%) were achieved with hydrophobic supports based on zirconium benzenephosphonate. The biocomposites can be stored for more than one month at 4 °C without loss of enzymatic activity, have been used in several cycles, and undergo limited thermal degradation when used at 40 °C.

Introduction Layered zirconium phosphates and phosphonates constitute a family of compounds that are very attractive for material chemists.1-3 R-Zirconium hydrogen phosphate, R-Zr(HPO4)2‚H2O, is considered the archetype of the family. Its structure arises by the packing of layers that consist of zirconium atoms lying in a plane and sandwiched by O3P-OH groups situated alternatively above and below the plane.4 The compound is highly insoluble in strong acidic solutions and undergoes limited hydrolytic attack in an alkaline medium. The acidic protons, 6.64 mmol/g, can be replaced by inorganic and organic cations. Moreover, protophilic organic and organometallic species can be intercalated in the interlayer region.1 Another very attractive aspect of zirconium phosphate chemistry is related to the possibility of replacing the P-OH groups anchored to the layer surface with P-OR or P-R groups without altering the inorganic texture of the layer (R is an organic group).3,5 The layered compounds so obtained are considered organic derivatives of the parent RZr(HPO4)2‚H2O, with the layers being functionalized with a large variety of organic groups (alkyl, aryl, carboxylic, amino acidic, sulfophenyl). It is also possible to change the hydrophobic/hydrophilic character of the layer surface * To whom correspondence should be addressed. E-mail: ucost@ unipg.it. Telephone: 075/5855565. Fax: 075/5855566. † Laboratorio di Chimica Organica. ‡ Laboratorio di Chimica Inorganica. (1) Alberti, G.; Casciola, M.; Costantino, U.; Vivani, R. Adv. Mater. 1996, 8, 291. (2) Clearfield, A.; Costantino, U. Layered Metal Phosphates and their Intercalation Chemistry. In Comprehensive Supramolecular Chemistry, Vol. 7; Alberti, G., Bein, T., Vol. Eds.; Pergamon: Elsevier Science Ltd Press: 1996; p 107. (3) Clearfield, A. Metal Phosphonate Chemistry. In Progress in Inorganic Chemistry, Vol. 47; Karlin, K. D., Ed.; Wiley & Sons: New York, 1997; pp 371-510. (4) Troup, J. M.; Clearfield, A. Inorg. Chem. 1977, 16, 3311. (5) Alberti, G.; Costantino, U.; Allulli, S.; Tomassini, N. J. Inorg. Nucl. Chem. 1978, 40, 1113.

by interspersing the inorganic P-OH groups with suitable organic groups.6 Many of these layered compounds give rise to exfoliation reactions with a consequent increase of the specific surface area.7,8 Thus, layered zirconium phosphates and phosphonates are a very versatile class of compounds for materials design. Examples of constructing materials useful in the fields of electrochemistry,9 photochemistry, nonlinear optics,10 and chiral recognition11 have been reported. Few papers describe the interaction of zirconium phosphates with bioactive materials. Rozie`re and coworkers12 reported the preparation of composites of R-Zr(HPO4)2‚H2O and proteins (protamine, gelatine, lysozyme). More recently, Kumar and Chaudary13 reported the immobilization of several proteins (myoglobin, lysozyme, hemoglobin, chymotripsin, glucose oxidase) at the interlayer region of zirconium phosphate and the denaturation and activation of myoglobin when intercalated into zirconium carboxyethanephosphonate.14 It is current opinion that surface adsorption is the method of immobilization which induces fewer modifications of the active conformation of the enzymes,15,16 especially when the solid surfaces are hydrophilic and/or (6) Alberti, G.; Costantino, U.; Kornyei, J.; Luciani, M. L. React. Polym. 1985, 4, 1. (7) Alberti, G.; Casciola, M.; Costantino, U. J. Colloid Interface Sci. 1985, 107, 256. (8) Alberti, G.; Casciola, M.; Costantino, U.; Peraio, A.; Rega, T. J. Mater. Chem. 1995, 5, 1809. (9) Casciola, M. See ref 2, Chapter 12. (10) Katz, H. E.; Wilson, W. L.; Sheller, G. J. Am. Chem. Soc. 1994, 116, 6636. (11) Cao, G.; Garcia, M. E.; Alcala`, M.; Burges, L. F.; Mallouk, T. E. J. Am. Chem. Soc. 1992, 114, 7574. (12) Ding, Y.; Jones, D. J.; Maireles-Torres, P.; Rozie`re, J. Chem. Mater. 1995, 7, 562. (13) Kumar, C. V.; Chaudhari, A. J. Am. Chem. Soc. 2000, 122, 830. (14) Kumar, C. V.; Chaudhari, A. Chem. Mater. 2001, 13, 238. (15) Ipson, A. P.; Dunnill, P.; Lilly, M. D. Biocatalysis 1990, 3, 329. (16) Znezevic, K.; Mojovic, L.; Adnadjevic, B. Enzyme Microb. Technol. 1998, 22, 275.

10.1021/la0262912 CCC: $22.00 © 2002 American Chemical Society Published on Web 10/10/2002

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hydrophobic, allowing the adsorbed enzyme to mimic the interactions of its natural environment.17 A large research program has been undertaken to immobilize proteins and enzymes on the surface of different layered phosphates and phosphonates and to select those most appropriate for a given protein in terms of stability and activity of the biocomposite obtained. Lipases were initially considered. These enzymes are useful in biocatalysis because of their capacity to catalyze the hydrolysis/synthesis of a wide range of soluble or insoluble carboxylic acid esters and amides, with high selectivity and/or stereospecificity.18-20 Most of the reactions described in the literature21,22 are catalyzed by crude preparations that may contain several isoforms. The presence of isoenzymes and/or isoforms with very slight differences in physical-chemical properties but with significant differences in activity and stereospecificity toward different substrates may reduce their usefulness in the preparation of chiral compounds in optically pure form. The usual methods of purification take a long time and are expensive. The biomaterial obtained is often unstable, and in some cases it is not possible to separate the various isoforms. For these reasons, it is very important to select supports able to separate and immobilize different isoforms and to improve the stability and activity of biomaterial. This paper describes new biocomposites obtained by supporting lipase from Candida rugosa (CRL) on zirconium phosphates and phosphonates. The supports have different surface areas and different hydrophobic/hydrophilic characters. The activity of immobilized lipase was assayed at various lipase loadings, while lipase stability was tested by a series of successive hydrolyses and after incubation at 40 °C. Experimental Section 1. Chemicals. ZrOCl2‚8H2O is a Merk “pro analysi” product. C6H5PO3H2 and HOOCC2H4PO3H2 are Fluka reagents. Tetraethylsilicate was an Aldrich product. Lipase from Candida rugosa (Crude CRL E.C.3.1.1.13 type VII) was supplied by Sigma Chemical Co., and Coomassie Brilliant Blue G-250, by Bio-Rad. All other chemicals used are C. Erba R.P.E reagents. 2. Preparation of Supports. Well-crystallized R-Zr(HPO4)2‚ H2O was obtained according to the method proposed by Alberti and Torraca.23 To a solution, prepared by dissolving 10.1 g of ZrOCl2‚8H2O in 160 mL of deionized water, were added 8.5 mL of hydrofluoric acid (40%) and 92 mL of H3PO4 (85%). The H3PO4 concentration was 5.2 M, and the molar ratios H3PO4/Zr and HF/Zr were 43 and 6.1, respectively. The obtained clear solution was heated at 80 °C for 4 days, maintaining the volume constant by continuous addition of water. Decomposition of zirconium fluoro-complexes permits the precipitation of a microcrystalline powder (average size 5 µm) of Zr(HPO4)2‚H2O. The HF procedure was also used to prepare the following phosphonates and phosphate-phosphonates: Zirconium benzenephosphonate: C6H5PO3H2 concentration 1 M; C6H5PO3H2/Zr and HF/Zr molar ratios 10 and 30, respectively.5 (17) Bosley, J. A.; Clayton, J. C. Biotechnol. Bioeng. 1994, 43, 934. (18) Santaniello, E.; Ferraboschi, P.; Grisenti, P.; Manzocchi, A. Chem. Rev. 1992, 92, 1071. (19) Cipiciani, A.; Fringuelli, F.; Mancini, V.; Piermatti, O.; Scappini, A. M. Tetrahedron 1997, 53, 11853. (20) Cipiciani, A.; Bellezza, F.; Fringuelli, F.; Stillitano, M. Tetrahedron Asymm. 1999, 10, 4599. (21) Tomizuka, N.; Ota, Y.; Yamada, K. Agr. Biol. Chem. 1966, 1090, 30576. (22) Veeragavan, K.; Gibbs, B. F. Biotechnol. Lett. 1989, 11, 779. (23) Alberti, G.; Torracca, E. J. Inorg. Nucl. Chem. 1968, 30, 317.

Letters Zirconium carboxyethanephosphonate: HOOCC2H4PO3H2 concentration 4 M; HOOCC2H4PO3H2/Zr and HF/Zr molar ratio 10 and 6, respectively.24 Zirconium benzenephosphonate phosphate: H3PO4 + C6H5PO3H2 concentration 6.8 M; C6H5PO3H2/C6H5PO3H2 + H3PO4, P/Zr, and HF/Zr molar ratios 0.01, 80, and 30, respectively.6 Zirconium carboxyethanephosphonate phosphate: HOOCC2H4PO3H2 + H3PO4 concentration 5 M; HOOCC2H4PO3H2/ HOOCC2H4PO3H2 + H3PO4, P/Zr, and HF/Zr molar ratios 0.33, 30, and 6, respectively.6 All the compounds were washed with deionized water until the pH of the washing water was about 4 and then air-dried. The X-ray powder diffraction (XRDP) patterns of the above-mentioned materials were in agreement with those already published.5,6,24 A colloidal dispersion of exfoliated zirconium phosphate was prepared by titrating 1 g of R-Zr(HPO4)2‚H2O, suspended in 68 mL of water with 32 mL of 0.10 M n-propylamine solution, at room temperature under vigorous stirring.7 Solid dispersions of silica and zirconium phosphate were prepared by adding 12 g of tetrapropylammonium oligosilicate solution8 (silica content: 15% w/w) to the above-mentioned colloidal dispersion of exfoliated zirconium phosphate. The treatment of the mixture with a few milliliters of 1 M acetic acid produced a sudden gelification. The gel was passed through a syringe to obtain a spaghetti-like shape, air-dried, and then calcined in an oxygen atmosphere at 650 °C, for at least 5 h.8 Solid dispersions of silica and zirconium phosphate benzenephosphonate were prepared by the same procedure, but the gel was first washed with HCl solution and then dried at 250 °C. 3. Characterization of the Supports. Zirconium phosphate and phosphonates as well as solid dispersions of silica and zirconium phosphate were characterized for their chemical composition, X-ray powder diffraction patterns, specific surface area, and surface ion exchange capacity. The molar ratio phosphate/benzenephosphonate or carboxyethanephosphonate in the solid was obtained by 31P NMR measurements, performed in solution, with a Brucker AC200 spectrometer, after dissolution of a weighed amount of sample (0.050 mg) in a few drops of concentrated HF and about 1 mL of DMSO-D6 as solvent.6 The X-ray powder diffraction (XRPD) patterns were taken with a computerized Philips PW1710 diffractometer using Cu KR radiation, operating at 40 kV and 20 mA, step scan 1° min-1. The specific surface areas were calculated according to the BET method from N2 adsorption isotherms taken with a computer controlled Micromeritics ASAP2010 unit, at the boiling temperature of liquid nitrogen. The surface ion exchange capacity was obtained by the method described in ref 25. 0.2 g of sample, previously dispersed in 25 mL of 0.2 M CsCl solution, was titrated, up to pH 6, with a 0.100 M CsOH solution by means a Radiometer automatic titrimeter. 4. Preparation of CRL Solution. Lipase from Candida rugosa was treated according to the following procedure: 3 g of lipase was dissolved in 30 mL of sodium phosphate buffer (25 mM, pH 7.2) at 4 °C. After 4 h of gentle stirring, the suspension was centrifuged at 3000 rpm for 30 min to remove the precipitate. The supernatant was dialyzed overnight against deionized water (2 per 2 L). The solution was stored at 4 °C, to limit possible thermal degradation of the protein. 5. Protein Assays of CRL Solution. The protein concentration of the solution was measured using the Bradford method.26 The calibration curve was obtained with BSA (bovine serum albumin) for concentrations from 0.1 to 1 mg/mL. 6. Free CRL Activity Assay. The hydrolytic activity of the protein solution was assayed by the rate of hydrolysis of p-nitrophenylacetate (p-NPA) to produce p-nitrophenol and acetic acid. In a typical experiment, 1 mL of p-NPA (0.1 M in CH3CN) was added to 10 mL of phosphate buffer (10 mM, pH 7.2). The mixture was stirred vigorously, and then an aliquot of lipase solution was added. As the hydrolysis proceeded, the released acetic acid was continuously titrated with a 0.02 M NaOH (24) Alberti, G.; Costantino, U.; Casciola, M.; Vivani, R.; Peraio, A. Solid State Ionics 1991, 46, 61. (25) Alberti, G.; Bernasconi, M. G.; Costantino, U.; Casciola, M. Ann. Chim. 1978, 68, 265. (26) Bradford, M. Anal. Biochem. 1976, 72, 248.

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Table 1. Physical-Chemical Properties of Layered Zirconium Phosphates and Phosphonates Used as CRL Supports composition

acronym

size

BET surface area (m2/g)

surface ion exchange capacity (µmol/g)

SiO2 R-Zr(HPO4)2 R-Zr(HPO4)2-SiO2 (36% R-ZrP by weight) R-Zr(PO3CH2CH2COOH)1.7 (HPO4)0.3 R-Zr(PO3CH2CH2COOH)2 R-Zr(C6H5PO3)1.62(HPO4)0.38 R-Zr(C6H5PO3)2

SiO2 R-ZrP R-ZrP-SiO2 R-ZrP-CEP R-ZrCEP R-ZrP-BP R-ZrBP

granules (0.5-2 mm) platelet (1-10 µm) granules (0.5-2 mm) platelet (1-10 µm) platelet (1-10 µm) platelet (1-10 µm) platelet (1-10 µm)

208 2.4 400 (120)a 3 3 8 8

5 800 72 85 10

a

Calculated surface area covered by ZrP lamellae (see text).

solution. This was done automatically using an Autotitrator Mettler Toledo DL21, operating at pH-stat mode and pH ) 7.2. One lipase unit (U) is defined as the amount of enzyme which is required to liberate 1 µmol of acetic acid/min under the assay conditions. 7. CRL Immobilization. 60 mg of solid support was equilibrated with several volumes of 0.5 mg/mL lipase solution in a sealed vessel and stirred for 16 h at 4 °C. The suspension was then centrifuged at 3000 rpm for 10 min to separate the supernatant and the solid. The biocomposite obtained was washed twice with a few milliliters of deionized water and then filtered. The supernatant was analyzed for the protein content, according to the Bradford method. The amount of bonded protein was determined indirectly by calculating the difference between the amount of protein introduced into the reaction mixture and the amount of protein found in the supernatant. The residual free activity of the supernatant solution was measured as described above. Lipase loading (U/g of support) was obtained by subtracting the residual free activity of the supernatant from the lipase activity before the immobilization procedure. 8. Immobilized CRL Activity Assay. One milliliter of p-NPA (0.1 M in CH3CN) was added to 10 mL of phosphate buffer (10 mM, pH 7.2). The mixture was stirred vigorously, and then 5 mg of the biocomposite was added. As hydrolysis proceeded, the acetic acid was titrated as described above. The activity of immobilized lipase was defined as the micromoles of acetic acid formed in 1 min by 1 g of biocomposite. The immobilization efficiency was then calculated from the ratio of the activity of the immobilized lipase to the lipase loading. 9. Stability Assays of Immobilized CRL. Either free (50 µL) or immobilized lipase (10 mg solid) was incubated in 10 mL of phosphate buffer (10 mM, pH 7.2) at 40 °C for from 2 to 24 h to determine thermal deactivation of immobilized enzyme. The residual activity was then assayed as previously described. 10. Immobilized CRL Reuse. 1 mmol of p-NPA (0.181 g) was added to 0.24 g of biocomposite previously dispersed in 10 mL of phosphate buffer (10 mM, pH 7.2), and the acid released was titrated as described above. The biocomposite was then removed from the reaction mixture by filtration, washed with distilled water, and reutilized for successive experiments.

Results and Discussion 1. Physical-Chemical Characterization of the Support. Chemical composition, size and shape, BET specific surface area, and, where appropriate, surface ion exchange capacity of materials used as supports of CRL are reported in Table 1. The supports are listed according to the increase of hydrophobic character of the functional groups and are identified by an acronym. A solid dispersion of zirconium phosphate in silica (R-ZrP-SiO2) possesses the highest surface area and surface ion exchange capacity. According to ref 8, the composite contains bilamellae of zirconium phosphate pyrophosphate chemically anchored to macroporous silica. The surface of the bilamella, schematically drawn in Figure 1, is a hexagonal array of P-OH groups, and there are four groups every 100 Å2.4 From this datum and the surface ion exchange capacity (0.8 mmol H+/g), it is possible to evaluate the extension of the specific surface area of the solid dispersion covered

Figure 1. Schematic representation of one side of zirconium phosphate and phosphonate lamella (R ) OH, C2H4COOH, C6H5).

Figure 2. Same lamella of Figure 1 containing two different groups (R ) OH; R′ ) C2H4COOH, C6H5).

by ZrP lamellae: 8 × 10-4 mol/g × 6.02 × 1023 mol-1 × 100/4 Å2 × 10-20 m2/Å2 ) 120 m2/g. The other supports have a much lower specific surface area and ion exchange capacity than R-ZrP-SiO2. However, the disposition of the groups lying on the surface is identical to that found on R-ZrP, the P-OH groups being partially or totally replaced by P-C6H5 or P-CH2CH2COOH (see Figures 1 and 2). It may be noted from Table 1 that the adsorbed enzyme may experience different interactions: ionic interactions arising as a consequence of proton transfer from POH groups of the support to external amino groups of the enzyme (i.e. protonation of lysine groups) and pure hydrophobic interactions with the P-C6H5 groups of R-ZrBP. 2. Immobilization Procedures and Loading. The adsorption of lipase as a function of time of equilibration and of the amount of protein added in solution was determined for the various supports, including macroporous silica. Figures 3 and 4 report typical results of the experiments performed. The former figure refers to the uptake of CRL as a function of time when 60 mg of R-ZrPBP is suspended, at 4 °C, in 4 mL of a solution containing 0.5 mg/mL of protein. The latter refers to the adsorption isotherms obtained by equilibrating, at 4 °C for 16 h, 60 mg of R-ZrBP or of R-ZrP-BP with increasing volumes of a solution containing 0.5 mg of CRL per mL. It may be seen that the achievement of saturation capacity requires about 16 h of equilibration. Moreover, the curves of Figure 4 indicate that in the initial stages of adsorption almost

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Table 2. Analytical Data of Preparation of Biocomposites Obtained by Equilibrating Different Supports with Increasing Volumes of CRL Solution (c ) 0.5 mg of protein/mL of Solution) support SiO2 R-ZrP R-ZrP-SiO2 R-ZrP-CEP R-ZrCEP R-ZrP-BP R-ZrBP a

mg of added protein/g of support

mg of bounded proteina/g of support

protein immobilization (%)

Q [(mg/g)/(mg/mL)]

Qn {[(mg/g)/(mg/mL)]/m2}

16.7 16.7 33.3 50.0 16.7 33.3 50.0 16.7 33.3 50.0 16.7 33.3 50.0 16.7 33.3 50.0 16.7 33.3 50.0

0.3 10.0 14.0 16.0 6.7 10.6 12.0 10.7 14.0 16.5 7.7 13.9 15.0 14.7 24.6 29.0 12.0 18.0 19.0

2 60 42 32 40 32 24 64 42 33 46 42 30 88 74 58 72 54 38

0.6 50.0 48.3 47.1 22.3 31.2 31.6 59.4 48.3 50.0 28.5 47.9 42.8 245.0 189.2 138.1 85.7 78.3 61.3

0.003 20.8 20.1 19.6 0.2 0.3 0.3 19.8 16.1 16.7 9.5 15.9 14.3 30.6 23.6 17.3 10.7 9.8 7.7

Calculated from the protein remaining in solution after immobilization (determined by the Bradford method).

Figure 3. Adsorption of CRL on R-ZrP-BP as a function of equilibration time (60 mg of support, 4 mL of solution 0.5 mg/ mL, T ) 4 °C).

Figure 4. CRL surface uptake of R-ZrP-BP and of R-ZrBP as a function of the amount of CRL added in solution (60 mg of support, lipase solution 0.5 mg/mL, T ) 4 °C).

all the lipase present in solution is uptaken by the support; then, as more protein is added, a plateau of uptake, indicating the saturation capacity, is reached. XRPD patterns of the solids, equilibrated with CRL solutions, did not show detectable expansion of the original interlayer distances, indicating that protein uptake mainly occurs on the surface of the microcrystals. The results obtained with the various supports are summarized in Table 2. It may be seen that the adsorption of lipase on calcined macroporous silica is very low. Zirconium phosphate, notwithstanding its low specific surface area, shows a relatively high protein uptake. However, in the presence of protein solution, the samples give rise to colloidal dispersions that make successive tests difficult. This phenomenon was not observed when the R-SiO2-ZrP composites and the other supports were equilibrated with the protein solution. Note that the extract of lipase from Candida rugosa is only partially

purified (see Experimental Section) and the protein solution still contains species from the original extract that may be intercalated and may exfoliate the R-ZrP microcrystals. Consequently, there is an increase of surface area that could account for the high value of protein uptake. The data in Table 2 indicate that the amount of protein adsorbed per gram of support seems to depend much more on the hydrophobic/hydrophilic character of the surface than on the surface area. For example, the amount of protein absorbed on R-ZrP-BP is more than three times that absorbed by R-ZrP-SiO2, even though its surface area is at least 10 times lower. Furthermore, the uptake on R-ZrP-BP is significantly higher than that observed on R-ZrBP even though the samples have very similar surface area. Table 2 also reports the value of a distribution quotient, Q, defined as the ratio between milligrams of protein adsorbed per gram of support and milligrams of protein per milliliter of solution at equilibrium. This parameter gives an indication of the affinity of the protein for the support. The distribution quotients were normalized to 1 m2 of surface (see column Qn), to compare supports with different surface areas. The Qn parameter reaches its maximum value for R-ZrP-BP. It seems that there is a cooperative positive effect on the adsorption of CRL, due to the simultaneous presence of protogenic and hydrophobic groups on the surface of the support. To test the possible desorption of lipase from the biocomposites, the materials were stirred in the reaction medium for 30 min. After filtration, the protein concentration in the elute was measured, and no significant traces of protein were observed. The same experiment was carried out in phosphate buffer (0.1 M, pH 7.2), and also in this case no significant desorption of protein was observed. The stability of derivatives upon washing indicates an adsorption rather than absorption of the protein on the various supports. 3. Hydrolytic Activity of the Biocomposites. The extract of CRL was only partially purified and could contain inactive proteins revealed by the Bradford method. Therefore, the loading of the biocomposites in terms of active enzyme was obtained by measuring the difference between the free lipase activity in solution before and after the equilibration with the different supports. The determination of the enzymatic activity was performed by measuring the amount of acetic acid produced in

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Table 3. Hydrolytic Activity of Candida rugosa Lipase Immobilized on Different Supports, Assayed in the Hydrolysis of p-NPA support R-ZrP-SiO2 R-ZrP-CEP R-ZrCEP R-ZrP-BP R-ZrBP a

Ua(free)/g of support

lipase loadingb (U(bounded)/g of support)

lipase immobilization (%)

activity (µmol/min‚g of catalyst)

immobilization efficiencyc (%)

321.7 643.3 965.1 643.3 1286.7 1930.0 828.3 1656.7 2483.3 500.0 1000.0 1500.0 908.3 1816.7 2724.9

90.1 141.5 173.7 283.0 360.3 463.2 248.3 314.8 273.1 300.0 410.0 450.0 327.0 472.3 599.5

28 22 18 44 28 24 30 19 11 60 41 30 36 26 22

2.0 1.4 1.7 100.0 116.7 120.4 65.0 85.0 100.0 223.3 205.0 181.7 311.7 235.0 330.0

2 1 1 35 32 26 26 27 36 74 50 40 95 50 55

Lipase unit (see Experimental Section). b [U(free) - U(residual)]/g of support. c (activity/lipase loading) × 100.

Figure 5. Hydrolytic activity of CRL supported on R-ZrP-BP as a function of lipase loading.

Figure 7. Thermal deactivation of CRL immobilized on R-ZrPBP and free CRL at 40 °C.

Figure 6. Catalytic efficiency of CRL supported on R-ZrP-BP as a function of lipase loading.

Figure 8. Effect of repeated assays on the hydrolytic activity of CRL immobilized on R-ZrP-BP.

solution by catalyzed hydrolysis of p-nitrophenylacetate, as described in the Experimental Section. Table 3 reports the lipase loading, the activity of the biocomposites, and the immobilization efficiency, defined as the ratio between the activity and the lipase loading. Immobilization of CRL on different supports leads to materials with different activities. High activities and efficiencies were observed for R-ZrP-BP and R-ZrBP, while the R-ZrP-SiO2 solid dispersion has a very low activity despite the good lipase loading. It is likely that the interactions of enzyme with this type of support produce a distortion of the enzymatic site that does not recognize the p-NPA substrate but could recognize other substrates. For all the investigated supports, an increase of adsorbed protein leads to derivatives with different activity. Figure 5 reports the hydrolytic activity of samples of R-ZrP-BP with an increasing lipase loading. Samples with increased amount of supported enzyme show a smaller activity. When the activity plot is converted into an efficiency plot, we obtained a similar trend, with smaller efficiencies at higher loading (see Figure 6). This effect could be related to a possible adsorption of new enzyme molecules onto the previously adsorbed ones,

to form “multilayers” or “clusters”. The increase of loading may not enhance the activity or efficiency of the biocatalyst because of steric hindrance to the diffusion of the substrate within the multilayer structure of the enzyme molecules. On the other hand, the biocomposites can be stored at 4 °C for more than one month without appreciable loss of catalytic activity. The stability of native and immobilized lipase was also studied after incubation at 40 °C. The plot residual activity versus incubation time is presented in Figure 7 for the R-ZrP-BP biocomposites. After 6 h of incubation, the enzyme still retains 50% of its original activity. The percentage of inactivation remains constant for longer periods of time. In the same conditions, free lipase shows an exponential decay, so that, after 24 h of incubation, the residual activity was lower than that of immobilized enzyme. This result suggests that the immobilization process enhances lipase thermal stability. Finally, the operational stability of the immobilized system was tested by reusing the biocomposite in successive hydrolyses. The data in Figure 8 show that the biocomposite retains 20% of the initial activity, even after five batches.

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It is difficult to comment on the loss in activity with the recycling process. However, possible release of supported lipase into solution may be excluded on the basis of the stability tests above-reported (see paragraph 2), and the loss in activity may be ascribed to progressive denaturation of supported enzyme and/or to loss of access of active sites for its conformational changes. 4. Conclusions. In this work layered zirconium phosphates and phosphonates with different functional groups, anchored to the inorganic backbone, were used as supports of lipase from Candida rugosa. The uptake capacity of the supports is strongly affected by the chemical character of the surface, increasing as the hydrophobic character of the functional groups increases. The catalytic efficiency of biocomposites, assayed in the hydrolysis of p-NPA, is generally good, and values as high as 95% were obtained with hydrophobic supports such as R-ZrP-BP and R-ZrBP, at low protein loading. Moreover, the recycle potential of the immobilized enzyme and operational stability at 40 °C were satisfactory.

Letters

Given the rich chemistry of metal phosphonates3 and the large variety of functional groups that may be present on their surface, it may be possible to select the support that offers interaction sites to the investigated protein without altering the active conformation. Following this line of research, the immobilization of other proteins on the chosen zirconium phosphonate has been undertaken. Our efforts are now directed to the study of the arrangement of lipase molecules on the surface of R-ZrP-BP and to exploring the possibility of selectively immobilizing different isoforms of lipases by exploiting the different characteristics of the supports. These biocomposites containing different isoforms could show different activity and enantioselectivity. Acknowledgment. The Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST) and Universita` degli Studi di Perugia (COFIN 2001, prot. N. 2001038157) are thanked for financial support. LA0262912