Smart Biocatalysts and Biocatalytic Hydrogels - American Chemical

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Bioconjugate Chem. 2001, 12, 301−306

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Enzyme Conjugation to the Polysaccharide Chitosan: Smart Biocatalysts and Biocatalytic Hydrogels Rafael Vazquez-Duhalt,† Raunel Tinoco,† Paul D’Antonio,‡ L. D. Timmie Topoleski,‡ and Gregory F. Payne*,§ Instituto de Biotecnologia UNAM, Apartado Postal 510-3, Cuernavaca, Morelos, 62250 Mexico, Department of Mechanical Engineering and Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250, and Center for Agricultural Biotechnology, 5115 Plant Sciences Building, University of Maryland, College Park, Maryland 20742. Received September 15, 2000; Revised Manuscript Received December 27, 2000

Laccase from Coriolopsis gallica was conjugated to the renewable biopolymer chitosan using carbodiimide chemistry. The laccase-chitosan conjugate was observed to offer three unique properties. First, the laccase-chitosan conjugate displayed pH-responsive behavior such that the conjugate was soluble and active under acidic conditions, but precipitated when the pH was raised toward neutrality. Second, the laccase-chitosan conjugate was more stable than free laccase at extreme pHs. At pH 1, the inactivation rate constant (kin) for the soluble laccase-chitosan conjugate was 20-fold less than that for free laccase. At pH 13, kin for the insoluble laccase-chitosan conjugate was nearly 3-fold less than that for free laccase. Finally, the laccase-chitosan conjugate could be cross-linked under mild conditions to create biocatalytic hydrogels. Potential benefits for enzyme-chitosan conjugates are discussed.

INTRODUCTION

There are a variety of reasons for coupling polymers with enzymes. Polymer conjugation has been reported to reduce the autolysis of proteolytic enzymes (1) and to allow enzymes to dissolve in nonaqueous solvents (2-4). “Smart” biocatalysts have been prepared by conjugating enzymes to stimuli-responsive polymers that respond to changes in pH (5, 6), ionic strength (7), temperature (810), redox potential (6), and light (11). Biocatalytic composites have been created by grafting olefinic functionality to enzymes and then incorporating these enzymemonomers into synthetic polymer maticies through standard free radical polymerization reactions (12, 13). Similarly, biocatalytic composites have been created with polyurethane-based (14-16) and silicon-based materials (17). Advantages of these biocatalytic composites are that the matrix can be selected to enhance performance (18) and the composites can be cast or formed into various shapes and sizes including foams (14-16) and monoliths (17). Finally, there are numerous examples in which enzymes bound to sugars, or sugar-based polymers are stabilized. Although the stabilization mechanism is not understood it may be due to; a reduction of autolysis (for proteases) (19, 20), multipoint attachment that may limit enzyme distortions (21, 22), or microenvironmental effects (23). Here we describe the use of chitosan as our polymer for enzyme conjugation. Chitosan is a biopolymer derived from chitin and, as shown in Figure 1, chitosan is made primarily of glucosamine repeating units. Because of its * To whom correspondence should be addressed. Phone: (301) 405-8389. Fax: (301) 314-9075. E-mail: [email protected]. † Instituto de Biotecnologia UNAM. ‡ Department of Mechanical Engineering. § Department of Chemical and Biochemical Engineering and Center for Agricultural Biotechnology.

high amino content, chitosan is soluble in mildly acidic aqueous solutions but is insoluble near and above its pKa of 6.3 (24, 25). As indicated in the upper path of Figure 1, we examined whether conjugation to chitosan could confer pH-responsive behavior to an enzyme. Additionally, chitosan’s amino groups are nucelophilic and reactive at higher pHs. The reactivity of these amino groups allows chitosan to be cross-linked under mild conditions to create gel matrixes (26) of various shapes and sizes including beads (27), membranes (28-30), and fibers (31-33). The lower path in Figure 1 shows that we examined whether cross-linking could be used to incorporate a conjugate into a chitosan hydrogel. It should be noted that cross-linked chitin and chitosan have been extensively studied as insoluble supports for enzyme immobilization (34, 35). As a model enzyme, we selected the fungal laccase from Coriolopsis gallica. Laccases are oxidative enzymes that function under acidic conditions (36, 37) and are actively being studied for applications in the pulp and paper industry (38) and for environmental remediation (39). The laccase from C. gallica has also been tested for the decolorization of textile effluents (40, 41), and has been observed to be capable of oxidizing polycyclic aromatic hydrocarbons (42). Because of the interest in laccase, several groups have examined its immobilization (43 and references therein) and one group recently reported the immobilization and cross-linking of laccase and chitosan (44). MATERIALS AND METHODS

Chitosan, 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS), and buffer salts were obtained from Sigma Chemicals Co. (St. Louis, MO). N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (ECD) was purchased from Fluka Chemie AG (Buchs, Switzerland). Laccase was obtained from the white-rot fungus C. gallica and purified as described previously (40).

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Figure 1. Enzyme-chitosan conjugate for pH-responsive solubility (upper path) and biocatalytic hydrogels (lower path).

The laccase-chitosan conjugate was prepared by mixing laccase (33 units) and chitosan (final concentration 1.0 w/w %) at pH 4.0 to obtain a solution with a molar ratio of glucosamine monomer/protein of 8000. To this solution, 1.6 mM of ECD was added (200 molar excess of relative to laccase). The reaction mixture was kept at room temperature for 2 h, and then the pH was increased to 8.0 by adding 10 volumes of 100 mM Tris buffer pH 8.0 and the suspension was cooled on ice. The precipitated laccase-chitosan conjugate was separated by centrifugation at 5000 rpm for 5 min. The pellet was then dissolved into 3 mL of 60 mM phosphate buffer and the pH was adjusted to 4.0 by adding 0.2 M HCl. When the laccase-chitosan conjugate was prepared for determining the pH-activity profile, for measuring stability at extreme pHs, and for preparing biocatalytic hydrogels, the preparation was precipitated and solubilized three additional times to remove nonconjugated laccase. The laccasechitosan conjugate was stored in solution (pH 4) at 4 °C. Enzymatic activities for free and conjugated laccase were determined colorimetrically using ABTS as the substrate. Assays were conducted in 1 mL reaction mixture containing 1 mM ABTS in a 60 mM acetate buffer (pH 4.5). Reactions were initiated by adding free or conjugated enzyme and laccase activity was estimated from the absorbance increase at 436 nm using an extinction coefficient of 23 900 M-1 cm-1 (45). Laccase units are defined as 1 µmol of substrate oxidized/min at 25 °C. The stability of free and conjugated laccase at extreme pHs was compared by incubating 100 µL of each preparation in buffered solutions of different pH. An acetateHCl buffer (60 mM) was used to achieve a pH of 1, while a sodium carbonate-NaOH buffer (60 mM) was used for pHs of 12 and 13. After incubation, the mixture was diluted to 1 mL with acetate buffer, pH 4.5, and enzyme activity was assayed at pH 4.5 as described above. The inactivation rate constants (kin) were determined from a first-order fit of the loss of enzyme activity over time. Biocatalytic gels were prepared by glutaraldehyde cross-linking. A solution containing laccase-chitosan conjugate (5.19 units/mL) was mixed with solution containing nonconjugated chitosan to obtain a solution with a laccase activity of 0.5 units/mL and a chitosan

concentration of 1 w/w %. To initiate cross-linking, differing amounts of a concentrated glutaraldehyde solution were added to these solutions to obtain glutaraldehyde concentrations between 3.1 and 31 mM (glutaraldehyde/glucosamine monomer molar ratios ranging from 0.05 to 0.5). Cross-linking was allowed to occur overnight at room temperature. The enzyme activity for the crosslinked chitosan gels could not be determined by the colorometric procedure because the gels absorbed the colored product of the ABTS reaction. To determine the laccase activity of the biocatalytic gels, the consumption of dissolved oxygen (Microelectronics Inc., Bedford, NH) was measured for the reaction of catechol (20 mM) in 60 mM acetate buffer, pH 4.5. The reactions were carried out in a sealed container and initiated by adding catechol. The relative stiffness (i.e., “strength”) of the crosslinked hydrogels was determined by a penetration method described by Gregson et al. (46). For this measurement, gels were formed in a cylindrical container (42.3 mm diameter × 9.0 mm high) by mixing varying amounts of concentrated glutaraldehyde to a chitosan solution (1 w/w %). The relative stiffness was determined by forcing a cylindrical probe (15.64 mm diameter) into the gels at a constant velocity of 0.1 mm/s. After correcting for the buoyancy, the measured force could be used to calculate an “apparent Young’s modulus” which is a relative measure of gel stiffness (46). RESULTS AND DISCUSSION

Purified laccase (33 units) from C. gallica was conjugated to chitosan using carbodiimide chemistry. Conjugation was performed at a pH of 4.0 and after conjugation, the solution pH was raised to 8 resulting in the formation of a white precipitate. The precipitate and supernatant were separated, and after adding phosphate buffer (pH 4) to the precipitate it was observed to redissolve in about 10 min. A sample of the redissolved precipitate was tested and observed to have 9.3 units of laccase activity. The unprecipitated, supernatant fraction was observed to have 16.3 units of laccase activity. This sample (designated cycle 1 in Figure 2) shows that 28% of the initial laccase activity was conjugated and the laccase-chitosan conjugate possess pH-responsive behavior.

Smart Biocatalysts and Biocatalytic Hydrogels

Figure 2. pH-responsive solubility of the laccase-chitosan conjugate. Laccase-chitosan conjugate was subjected to sequential cycles of precipitation at pH 8 and solubilization at pH 4. The activity of the precipitated (conjugate) and soluble (free laccase) fractions were measured under standard conditions (pH 4.5) after allowing about 10 min for the conjugate to redissolve. Cycle 1 represents the conjugation step and the soluble laccase from cycle 1 includes the activity of nonconjugated laccase. Precipitation can completely recover laccase activity of the conjugate.

Figure 3. pH-activity profile for free laccase and the laccasechitosan conjugate. Activity was measured colorometrically using ABTS as substrate. Conjugation does not affect laccase’s pH-activity profile.

To test the reversibility and completeness of precipitation, we subjected the laccase-chitosan conjugate to several cycles of precipitation (pH 8) and redissolution (pH 4). In each cycle, the precipitate was separated from the supernatant and redissolved in fresh buffer. Figure 2 shows that over the course of 10 cycles only small amounts of laccase activity were lost in the supernatants. More importantly, Figure 2 shows that the activity of the laccase-chitosan conjugate was almost quantitatively recovered by precipitation. Further, these results indicate that precipitation does not inactivate laccase. We measured the activity of free and conjugated laccases at various pHs to determine if conjugation affects the pH-activity profile. Figure 3 shows that laccase is active at pH 2 and the activity decreases monotonically to zero at neutral pHs. This pH-activity profile is similar to that reported by Xu (36) for the ABTS substrate. Figure 3 shows that conjugation to chitosan does not alter the pH-activity profile of laccase. Similar results were reported by D’Annibale et al. (44) for laccases that had been immobilized onto insoluble chitosan supports. The effect of conjugation on enzyme stability was compared by exposing free and conjugated enzyme to pH extremes. At various times, samples from extreme pH solutions were assayed for laccase activity under stan-

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Figure 4. Stabilization of laccase to extreme acid conditions. Enzyme was incubated for varying times at pH 1 and activities were measured under standard conditions (pH 4.5). Conjugation resulted in a 20-fold reduction in the inactivation rate constant (kin was 85 h-1 for free laccase and 3.6 h-1 for the laccasechitosan conjugate).

Figure 5. Stabilization of laccase to extreme base conditions. Enzyme was incubated for varying times at pH 12 or 13, and activities were measured under standard conditions (pH 4.5). The laccase-chitosan conjugate was fully stable at pH 12. Conjugation resulted in nearly a 3-fold reduction in the inactivation rate constant at pH 13 (kin was 4.5 h-1 for free laccase and 1.6 h-1 for the laccase-chitosan conjugate).

dard conditions (pH 4.5). Figure 4 shows that exposure to pH 1 leads to a rapid loss of activity for free laccase, while the soluble laccase-chitosan conjugate loses activity more slowly. The first-order inactivation rate constants (kin) for this extreme acid condition were estimated to be 85 and 3.6 h-1 for free and conjugated laccases, respectively. Figure 5 shows results for the alkaline extremes. At pH 12, the free enzyme was gradually inactivated (kin ) 0.89 h-1), while no inactivation could be detected for the insoluble laccase-chitosan conjugate. The protective effect of chitosan persisted even when the laccase-chitosan conjugate was incubated at pH 12 and 3 M NaCl (data not shown). At the extreme pH of 13, Figure 5 shows that the free laccase was inactivated more rapidly (kin ) 4.5 h-1) than the laccase-chitosan conjugate (kin ) 1.6 h-1). Fungal laccases have relatively good pH stabilities (47) and the results in Figures 4 and 5 show that conjugation to chitosan improves stabilities under acidic and basic pH extremes. This stabilization was observed with both soluble (low pH) and insoluble (high pH) conjugate. D’Annibale et al. (44) also observed that laccases that had been immobilized onto insoluble chitosan supports were more stable to acidic conditions. The nature of this stabilization is not known although various investigators

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Figure 6. Biocatalytic hydrogels created by glutaraldehyde cross-linking. Laccase-chitosan conjugate was mixed with nonconjugated chitosan (final laccase activity 0.5 units/mL and final chitosan concentration 1 w/w %) and cross-linked with varying levels of glutaraldehye (a molar ratio of glutaraldehyde to glucosamine monomer of 0.5 corresponds to the use of 32 mM glutaraldehyde). The apparent Young’s modulus was measured by a penetration method (46) while laccase activity was measured from dissolved oxygen consumption with catechol as the substrate.

have observed that sugars and sugar-based polymers can stabilize enzymes during lyophilization (23) and in nonaqueous environments (19, 20). Also, stabilization has been reported when enzymes are bound through multiple points (21, 22). Conceivably, the laccase may be covalently conjugated to chitosan at more than one “point”. Also, since laccases have a large number of surface carboxylates (48), it is possible that electrostatic interactions with chitosan’s amino groups could lead to multipoint, stabilizing interactions. Interestingly, when we “conjugated” laccase to glucosamine (the monomeric unit of chitosan), we observed no stabilization of laccase (data not shown). Laccase-containing chitosan hydrogels were prepared by glutaraldehyde cross-linking. To prepare these hydrogels, laccase-chitosan conjugate was diluted with nonconjugated chitosan (final polymer concentration 1 w/w % and final laccase activity 0.5 units/mL), and varying levels of glutaraldehyde were added to these solutions (pH 4). Over the course of several hours, these solutions were observed to form gels. As expected, Figure 6 shows that the stiffness, or apparent Young’s modulus, of the cross-linked chitosan gels increased with increasing glutaraldehye levels. The enzymatic activities of glutaraldehyde-cross-linked chitosan hydrogels were measured by adding 100 µL of gel to substrate solution (1 mL total volume) and measuring consumption of dissolved oxygen. Figure 6 shows that laccase activity was unaffected by cross-linking when less than 9 mM glutaraldehyde was used (0.45 molar ratios) laccase activity was completely lost. The results in Figure 6 indicate that glutaraldehyde can be used to create laccase-containing chitosan hydrogels and the crosslinker level must be selected to balance the need to retain enzymatic activity with the desire to achieve a given gel strength. It should be noted that this compromise is not limited to conditions examined in Figure 6, as alternative chitosan concentrations and different cross-linkers could also be considered. One additional characteristic of chitosan that must be considered, is that the amino groups may react with

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electrophilic substrates or products. Chitosan’s reactivity may limit its use in some applications, but it also provides additional opportunities. For instance, tyrosinase-containing chitosan gels can convert phenols into electrophilic quinones that react with the chitosan matrix (49). Because the substrate specificity of tyrosinase confers selectivity to the conversion and the quinone-chitosan reaction results in strong binding, tyrosinase and chitosan can be used to selectively remove phenolic contaminants from intermediate chemical process streams for industrial waste minimization (50-53). In conclusion, chitosan is a versatile biopolymer that is obtained from renewable resources and is readily biodegraded in the environment. We report three useful features that result from conjugating enzymes to chitosan. First, the laccase-chitosan conjugate has pH responsive behavior such that the conjugate is soluble at pHs of maximal laccase activity, yet insoluble at neutral pHs. This pH-responsive behavior endows the conjugate with the mass transfer advantages of soluble enzymes, while allowing the enzyme to be readily recoverable, the characteristic benefit of immobilized enzymes. Second, conjugation to chitosan stabilized laccase to extreme pHs. This stabilization occurred with soluble (low pH) and precipitated (high pH) conjugate. Third, the enzymechitosan conjugate can be cross-linked under mild conditions to yield biocatalytic hydrogels. This result suggests the potential for cross-linking biocatalytic hydrogels in various shapes and sizes including beads, membranes, fibers, and monolith coatings. ACKNOWLEDGMENT

Financial support for this research was provided by grants from Venture Innovations (as a subcontract to NSF Grant DMI-9901868), the Arthritis Foundation, and CONACYT for support of R.V.-D.’s sabbatical visit. The authors also appreciate technical assistance from Dr. Oleg Vesnovsky in the UMBC Laboratory for Implantable Materials. LITERATURE CITED (1) Yang, Z., Williams, D., and Russell, A. J. (1995) Synthesis of protein-containing polymers in organic solvents. Biotechnol. Bioeng. 45, 10-17. (2) Takahashi, K., Ajima, A., Yoshimoto, T., Okada, M., Matsushima, A., Tamaura, Y., and Inada, Y. (1985) Chemical reactions by poly(ethylene glycol) modified enzymes in chlorinated hydrocarbons. J. Org. Chem. 50, 3414-3415. (3) Ito, Y., Fujii, H., and Imanishi Y. (1993) Catalytic peptide synthesis by trypsin modified with polystyrene in chloroform. Biotechnol. Prog. 9, 128-130. (4) Inada, Y., Furukawa, M., Sasaki, H., Kodera, Y., Hiroto, M., Nishimura, H., and Matsushima, A. (1995) Biomedical and biotechnological applications of PEG- and PM-modified proteins. Trends Biotechnol. 13, 86-91. (5) Charles, M., Coughlin, R. W., and Hasselberger, F. X. (1974) Soluble-insoluble enzyme catalysis. Biotechnol. Bioeng. 16, 1553-1556. (6) Ito, Y., Kotoura M., Chung, D.-J., and Imanishi Y. (1993) Trypsin modification by vinyl polymers with variable solubilities in response to external signals. Bioconjugate Chem. 4, 358-361. (7) Hosino, K., Taniguchi, M., Marumoto, H., and Fujii, M. (1989) Repeated batch conversion of raw starch to ethanol using amylase immobilized on a reversible soluble-autoprecipitating carrier and flocculating yeast cells. Agric. Biol. Chem. 53, 1961-1967. (8) Hosino, K., Katagiri, M., Taniguchi, M., Sasakura, T., and Fujii, M. (1994) Hydrolysis of starch materials by repeated use of amylase immobilized on a novel thermo-responsive polymer. J. Ferment. Bioeng. 77, 407-412.

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