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Communications Cross-Linked Aggregates of Multimeric Enzymes: A Simple and Efficient Methodology To Stabilize Their Quaternary Structure Lorena Wilson, Lorena Betancor, Gloria Ferna´ ndez-Lorente, Manuel Fuentes, Aurelio Hidalgo, Jose´ M. Guisa´ n,* Benevides C. C. Pessela,* and Roberto Ferna´ ndez-Lafuente Department of Biocatalysts, Institute of Catalysis, CSIC, Madrid, Spain 28049 Received December 15, 2003; Revised Manuscript Received March 9, 2004
In this manuscript, we show that the immobilization of proteins following the technique of cross-linked protein aggregates (CLEAS) may permit the stabilization of the most complex multimeric enzymes by preventing their dissociation. To illustrate that, we have first prepared CLEAS with two tetrameric catalases. Activity recovery was over 40%, and no protein subunit could be desorbed from the CLEAS after boiling in SDS. More interestingly, the enzyme stability, which in its soluble form strongly depends on the enzyme concentration, becomes fully independent of this parameter. This permitted the enzyme stability to greatly increase under diluted conditions. In fact, diluted CLEAs presented a higher stability than those of their glyoxyl derivatives counterparts, which were unable to fully stabilize the multimeric structure of these tetrameric enzymes 1. Introduction The advances in microbiology and enzymology make available more and more complex enzymes with high interest for the organic chemist. This means that the solution to the stability problems of structurally complex enzymes has a growing relevance. One interesting example of these complex enzymes is the case of multimeric enzymes. Inactivation of these enzymes is commonly initiated by the dissociation of subunits. In fact, it is assumed that multipoint noncovalent assembly between monomers could stabilize the threedimensional structure of each individual monomer correctly assembled in the multimer.1-5 On the contrary, conformational changes promoted by any denaturing agent (heat, pH, organic solvents) on the small fraction of dissociated monomers (no stabilized by these interactions) could be much more rapid and intense.2,3 Dissociation of subunits may become even more relevant when working at an industrial scale under very mild experimental conditions, as a small fraction of subunits per reaction cycle could promote a very rapid deactivation of the enzyme bio-reactor. At first glance, we may assume that stabilization of the quaternary structure of multimeric enzymes may have very profitable effects on their industrial performance. Immobilization of multimeric enzymes on preexisting supports may be not enough to achieve this stabilization. It has been shown that the multisubunit immobilization plus * To whom correspondence should be addressed. E-mail: benicosta@ icp.csic.es (B.C.C.P.);
[email protected] (J.M.G.). Fax: 34-91-5854760. Phone: 91-585-4809.
further cross-linking may be enough to stabilize any multimeric structure.6-10 However, a new technique of enzyme immobilization via their aggregation and further cross-linking with different bifunctional reagents has been recently reported by Sheldon and co-workers. These preparations are called cross-linked enzyme aggregates (CLEAs).11-16 In our opinion, to the many advantages that this process has already exhibited (improved stability, selectivity and specificity compared to soluble enzymes), we add a critical point: this kind of enzyme immobilization should stabilize the quaternary structure of any multimeric enzyme, even better that multisubunit covalent attachment in preexisting supports, mainly for complex enzymes. Here, we have analyzed the performance of this methodology to stabilize the quaternary structure of two multimeric catalases. 2. Materials and Methods 2.1. Materials. Catalases from bovine liver (BLC) and Micrococcus lysodeikticus (MLC), diethyleneglycol-dimethyl ether and sodium periodate were purchased from Fluka (Buchs, Switzerland). Hydrogen peroxide, ethyleneglycol dimethyl ether, sodium borohydride, methanol, and glutaraldehyde were from Sigma Chemical Co (St. Louis, Missouri, USA). Poly(ethylene glycol) (PEG) 600 and 6000 was purchased from Merck (Schulchardt, Germany). Beads of 6% cross-linked glyoxyl-agarose gels were a gift from Hispanagar S. A. (Burgos, Spain) prepared as previously
10.1021/bm034528i CCC: $27.50 © 2004 American Chemical Society Published on Web 04/08/2004
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Figure 1. SDS-PAGE analysis of the proteins released from different preparations of Micrococcus lysodeikticus and bovine liver catalases. Lane 1: Molecular weight marker. Lane 2: Soluble catalase from Micrococcus lysodeikticus. Lane 3: Non reduced glyoxyl-agarose derivative of catalase from Micrococcus lysodeikticus Lane 4: Reduced glyoxyl agarose- catalase derivative. Lane 5: CLEA of catalase from Micrococcus lysodeikticus. Lane 6: Soluble bovine liver catalase. Lane 7: Non reduced glyoxyl-agarose derivative of catalase from bovine liver. Lane 8: Glyoxyl-agarose derivative of catalase from bovine liver. Lane 9: CLEA of catalase from bovine liver.
described.17 Immobilization in these supports was performed as previously described.10 All other reagents were of analytical grade. All experiments were performed, at least, by triplicate and error was never over 10%. 2.2. Determination of Enzyme Activity. Catalase activity was determined spectrophotometrically at 240 nm following the decomposition of 0.18% (w/w) hydrogen peroxide in 25 mM phosphate buffer, pH 7.5, 25 °C, using a molar absorption coefficient of 39.4 M-1 cm-1.18 0.1 mL of 30% H2O2 was added to 50 mL of 50 mM sodium phosphate buffer pH 7.0. To start the reaction, 0.02 mL of enzyme solution or CLEAs suspension were added. All of the measurements were carried out at room temperature. One catalase unit was defined as the amount of enzyme that decomposes 1 µmol of hydrogen peroxide per minute under the previously described conditions. For kinetic studies, the concentration ranged from 0 to 50 mM. 2.3. Determination of Protein Concentration. The amount of protein was determined as described by Bradford19 using bovine serum albumin as standard. 2.4. Preparation of CLEA-Catalase. CLEAs were prepared by slowly adding 30 mL of diethylene-glycoldimethyl ether to 10 mL of either (BLC) in 25 mM sodium phosphate buffer pH 7.0 under gentle stirring in an ice bath reactor, bovine liver catalase (605 900 UI/mL) or Micrococcus lysodeikticus (MLC), (442 500 UI/mL). The physically aggregated catalases were subjected to chemical crosslinking using glutaraldehyde, 5% v/v. The mixture was allowed to react for 1 h at room temperature. Finally, the whole reaction volume was duplicated with 0.1 M sodium
bicarbonate buffer pH 10, and during 30 min, 1 mg/mL of NaBH4 was added at intervals of 15 min. The CLEAs were then collected by centrifugation and washed thoroughly four times with 100 mM sodium phosphate buffer pH 7.0. (10 mL each time). The solid CLEAs were dispersed in 5 mL of 50 mM sodium phosphate buffer at pH 7.0 and stored at 4°C before use.11 2.5. Determination of the Enzyme Preparations Stability. Soluble enzyme and CLEAs (about 10000 UI/mL), were incubated at different temperatures (60 and 70 °C) in 50 mM sodium phosphate buffer at pH 7.0. Periodically, samples were withdrawn, and their remaining activities were assayed as described above. Stabilization was calculated as the ratio between the half-lives of the soluble proteins and the CLEAs preparations. Structural stabilization was determined by SDSanalysis of the supernatants achieved after boiling the different enzyme preparations.20 3. Results and Discussion 3.1. Kinetic Characterization of the CLEAs. CLEAs prepared with the catalase from Micrococcus lysodeikticus expressed about 40% of the offered activity and the one from bovine liver expressed 45%. In the case of the catalase from bovine liver, the Km increased from 30 mM ( 3 mM to 80 mM ( 9, very likely due to diffusion limitations. The Km of soluble catalase from Micrococcus lysodeikticus was over 100 mM,21 and an accurate determination of the value of the immobilized enzyme was very difficult. 3.2. Structural Stabilization of the Quaternary Structure of Multimeric Catalases. Figure 1 shows the SDS
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one subunit not bonded to the support, which kept less than 20% of the activity after a similar time period. 4. Conclusions
Figure 2. Thermal inactivation of CLEAs from bovine liver catalase. Enzyme suspensions were incubated at 60 °C in 50 mM sodium phosphate buffer pH 7.0. Further details are described in the Materials and Methods. b, CLEA (1000 U/mL); 9, CLEA (100 U/mL); 2, soluble (1000 U/mL); (, soluble (100 U/ mL). ×, glyoxyl derivative (100 U/mL).
PAGE of the supernatants obtained after boiling soluble catalases, CLEAs, and glyoxyl agarose catalase derivatives. Lanes 4 and 8 show that both bovine liver and Micrococcus lysodeikticus catalases immobilized on highly activated glyoxyl agarose released some protein subunits during boiling in the presence of SDS. The comparison between reduced and nonreduced glyoxyl derivatives gave an idea of the percentage of enzyme desorbed from these derivatives. Considering that the intensity of the band of the reduced derivative corresponds to 25-30% of that of the nonreduced one, we can assume that this support was able to bind 7075% of the enzyme subunits, leaving as an average value one subunit not directly bonded to the support. However, when these enzymes were aggregated and chemically cross-linked with glutaraldehyde, no enzyme subunits were detected in the supernatants when boiled in the presence of SDS (lanes 5 and 9). In fact, similar results could be achieved using crude protein extracts from a different origin, suggesting that the stabilization of the quaternary structure of any of their components have been achieved (results not shown). Therefore, the quaternary structure of catalases from Micrococcus lysodeikticus and bovine liver were completely stabilized by this treatment and proved to be more effective that immobilization on glyoxyl agarose. 3.3. Thermal Stability. Figure 2 shows the thermal inactivation course of soluble bovine liver catalase (BLC) and the CLEAs prepared with this enzyme (BLC-CLEAs) at different concentrations. It can be seen that the soluble enzyme is inactivated more rapidly than the BLC-CLEAs and that the inactivation rate depends on the enzyme concentration. However, in the case of the BLC-CLEAs, concentrated and diluted preparations have similar stability. In fact, diluted glyoxyl derivatives from bovine catalase were significantly less stable that the corresponding CLEA. The preparation of Micrococcus lysodeikticus CLEAs also permitted a significant increment of the thermo stability of this enzyme. Moreover, dilution of the CLEAs has no effect on the enzyme stability, whereas the soluble form stability strongly depends on the enzyme concentration. This stabilization allowed for the fact that the CLEA from Micrococus lysodeikticus catalase was much more stable than the soluble enzyme since it kept almost 50% of the residual activity after 30 min at 70 °C. Again, these results were much better that those achieved using diluted glyoxyl derivatives, with at least
The preparation of CLEAs has revealed itself as a powerful technique to solve one of the most important problems in using multimeric enzymes as biocatalyst: the stabilization of the quaternary structure in a very simple way. This may prevent enzyme inactivation, contamination of the reaction medium by enzymes, etc., without the cost of supports or sophisticated methodologies, and open new possibilities to the use of these complex enzymes as industrial biocatalysts. Moreover, the technique is readily amenable to scale-up and could be universally applicable, because it does not require the time and labor-intensive process of enzyme crystallization. The use of CLEAs may present some problems, mainly if the substrate is large. However, when used with small substrates, mainly with substrates not very suitable for the enzyme activity, it is possible to use this strategy with good results.11-16,22,23 In our model enzymes, catalases, we should consider that one of their more interesting used may be by co-aggregation with oxidases,10,24 and in these cases, the diffusion of the hydrogen peroxide should be not a real problem. Acknowledgment. We thank Hispanagar S.A. (Burgos, Spain) for their generous supply glyoxyl agarose gels and AECI for a fellowship for L.W. We gratefully recognize the support given by the Program of International Cooperation CSIC (Spain) - CONICYT (Chile). This work has been funded by the Spanish CICYT (BIO2001-2259 and -PPQ 2002-01231). The help and suggestions by MSc. AÄ ngel Berenguer (Departamento de Quı´mica Inorga´nica, Universidad de Alicante) are gratefully acknowledged. References and Notes (1) Ghadermarzi, M.; Moosavi-Movahedi, A. Influence of different types of effectors on the kinetics parameters of suicide inactivation of catalase by hydrogen peroxide. Biochim. Biophys. Acta 1999, 1431, 30-36. (2) Poltorak, O. M.; Chukhray, E. S.; Torshin, I. Y. Dissociative thermal inactivation, stability and activity of oligomeric enzymes. Biochem.Moscow 1998, 63, 360-369. (3) Poltorak, O. M.; Chukhray, E. S.; Torshin, I. Y.; Atyaksheva, L. F.; Trevom, M. D.; Chaplin, M. F. Catalytic properties, stability and the structure of the conformational lock in the alkaline phosphate from Escherichia coli. J. Mol. Catal. B: Enzym. 1999, 7, 181-172. (4) Attwood, P. V.; Geeves, M. A. Changes in catalytic activity and association state of pyruvate carboxylase which are dependent on enzyme concentration. Arch. Biochem. Biophys. 2002, 401, 63-72. (5) Mozhaev, V. V.; Klibanov, A. M.; Goldmacher, V. S.; Berezin, I. V. Operational stability of copolymerized enzymes at elevated temperatures. Biotechnol. Bioeng. 1990, 25, 1937-1945. (6) Fernandez-Lafuente, R.; Rodrigues, V.; Mateo, C.; Penzol, G.; Hernandez-Justiz, O.; Irazoqui, G.; Villarin˜o, A.; Ovsejevi, K.; Batista, F.; Guisa´n, J. M. Stabilization of multimeric enzymes via immobilization and post-immobilization techniques. J. Mol. Catal. B: Enzym. 1999, 7, 181-189. (7) Ferna´ndez-Lafuente, R.; Herna´ndez-Ruiz, O.; Mateo, C.; Terreni, M.; Alonso, J.; Garcı´a-Lo´pez, J.; Moreno, M. A.; Guisan, J. M. Stabilization of a tetrameric enzyme (R-amino acid ester hydrolase from Acetobacter turbidans, enables a very improved performance of ampicillin synthesis. J. Mol. Catal. B: Enzym. 2001, 11, 633-638.
Communications (8) Guisa´n, J. M.; Bastida, A.; Blanco, R. M.; Ferna´ndez-Lafuente, R.; Garcı´a-Junceda, E. Immobilization of enzymes by chemical modification with polyfunctional macromolecules. Immobilization of enzymes and cells. In Methods in Biotechnology; Bickerstaff, G., Ed.; The Humana Press: Totowa, NJ, 1997; pp 261-275. (9) Guisan, J. M.; Rodriguez, V.; Rosell, C. M.; Soler, G.; Bastida, A.; Blanco, R. M.; Ferna´ndez-Lafuente, R.; Garcı´a Junceda, E. Stabilization of immobilized enzymes by chemical modification with polyfunctional macromolecules. In Immobilization of enzymes and cells. In Methods in Biotechnology; Bickerstaff, G., Ed.; The Humana Press: Totowa, NJ, 1997; pp 289-298. (10) Betancor, L.; Hidalgo, A.; Fernandez-Lorente, G.; Mateo, C.; Fenandez-Lafuente, R.; Guisan, J. M. Preparation of a stable biocatalyst of Bovine Liver catalase using immobilization techniques. Biotechnol. Prog. 2003, 19, 763-767. (11) Cao, L.; Van Rantwijk, F.; Sheldon, R. Cross-Linked Enzyme Aggregates: A Simple and Effective Method for the Immobilization of Penicillin Acylase. Org. Lett. 2000, 2, 1361-1364. (12) Cao, L.; Van Langen, F.; Van Rantwijk, F.; Sheldon, R. Cross-linked aggregates of penicillin acylase: robust catalysts for the synthesis of B-lactam antibiotics. J. Mol. Catal. B: Enzym. 2001, 11, 665-670. (13) Asano, Y. Overview of screening for new microbial catalysts and their uses in organic synthesis- selection and optimization of biocatalysts. J. Biotechnol. 2002, 94, 65-72. (14) Lozinsky, V. I.; Plieva, F. M. Poly (vinyll alcohol) cryogels employed as matrixes for cell immobilization. 3. Overview of recent research and developments. Enzyme Microb. Technol. 1998, 23, 224-242. (15) Noritomi, H.; Koyama, K.; Kato, S.; Nagahama, K. Increased thermostability of cross-linked enzyme crystals of subtilisin in organic solvents. Biotechnol. Technol. 1998, 12, 467-469. (16) van Rantwijk, F.; Lau, R. M.; Sheldon, R. A. Biocatalytic transformation in ionic liquids. Trends Biotechnol. 2003, 21, 131-138.
Biomacromolecules, Vol. 5, No. 3, 2004 817 (17) Guisa´n, J. M. Aldehyde-agarose gels as activated supports for immobilization-stabilization of enzymes. Enzyme Microb. Technol. 1988, 10, 375-382. (18) Nelson, D. P.; Kiesow, L. A. Enthalpy of decomposition of hydrogen peroxide by catalase at 25 °C (with molar extinction coefficients of H2O2 solutions in the UV). Anal. Biochem. 1972, 49, 474-478. (19) Bradford, M. M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 1976, 72, 248-254. (20) Bastida, A.; Sabuquillo, P.; Armisen, P.; Ferna´ndez-Lafuente, R.; Huguet, J.; Guisan, J. M. A single sep purification, immobilization and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic support. Biotechnol. Bioeng. 1998, 58, 486-493. (21) Switala, J.; Loewen, P. C. Diversity of properties among catalases. Arch. Biochem. Biphys. 2002, 401, 145-154. (22) Wilson, L.; Illanes, A.; Pessela, B. C. C; Abian, O.; Ferna´ndezLafuente, R.; Guisa´n, J. M. Encapsulation of Cross-Linked Penicillin G Acylase Aggregates in LentiKats. Evaluation of a novel biocatalyst in organic media” Biotechnol. Bioeng. In press. (23) Wilson, L.; Illanes, A.; Abia´n, O.; Pessela, B. C. C.; Ferna´ndezLafuente, R.; Guisa´n J. M. Co-aggregation of penicillin g acylase and polyionic polymers: a simple methodology to prepare enzyme biocatalysts stable in organic media. Biomacromolecules In press. (24) Hidalgo, A.; Betancor, L.; Lo´pez-Gallego F.; Moreno, R.; Berenguer, J.; Ferna´ndez-Lafuente, R.; Guisa´n, J. M. “Preparation of a versatile biocatalyst of immobilized and stabilized catalase from Thermus thermophilus”. Enzyme Microb. Technol. 2003, 33, 278-285.
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