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Interaction and complex formation between catalase and cationic polyelectrolytes: Chitosan and Eudragit E100
International Journal of Biological Macromolecules 45 (2009) 103–108
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Interaction and complex formation between catalase and cationic polyelectrolytes: Chitosan and Eudragit E100 Valeria Boeris, Diana Romanini, Beatriz Farruggia ∗ , Guillemo Picó Bioseparation Lab. Chemical – Physics Department, Faculty of Biochemical and Pharmaceutical Sciences, CONICET, FonCyT and CIUNR, National University of Rosario, Suipacha 570 (S2002RLK) Rosario, Argentina
a r t i c l e
i n f o
Article history: Received 19 March 2009 Received in revised form 16 April 2009 Accepted 18 April 2009 Available online 3 May 2009 Keywords: Catalase Chitosan Eudragit E100 Polyelectrolyte
a b s t r a c t Interactions between catalase and the cationic polyelectrolytes: chitosan and Eudragit E100 have been investigated owing to their scientific and technological importance. These interactions have been characterized by turbidimetry, circular dichroism and fluorescence spectroscopy. It was found that the catalase conformation does not change significantly during the chain entanglements between the protein and the polyelectrolytes. The effects of pH, ionic strength and anions which modify the water structure were evaluated on the polymer–protein complex formation. A net coulombic interaction force between them was found since the insoluble complex formation decreased after the NaCl addition. Both polymers were found to precipitate around 80% of the protein in solution. No modification of the tertiary and secondary protein structure or the enzymatic activity was observed when the precipitate was dissolved by changing the pH of the medium. Chitosan and Eudragit E100 proved to be a useful framework to isolate catalase or proteins with a slightly acid isoelectrical pH by means of precipitation. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Chitosan is a cationic linear polymer, which is the second most abundant polymer in nature after cellulose [1]. Chitin is the primary structural component of the outer skeletons of crustaceans and it is also found in many other species such as mollusks, insects and fungi. The most commonly obtained form of chitosan is the ␣chitosan from crustacean chitin taken from crab and shrimp shell wastes. Chitosan has a wide variety of commercial and biomedical applications which are related to the physical properties of the biopolymer. Eudragit E100 is a cationic copolymer based on dimethylaminoethyl methacrylate and neutral methacrylic esters whose average molecular mass is 135.0000. It is highly used in pharmaceutical industry for immobilization and drug delivery [2]. Chitosan and Eudargit E100 exhibit a pH-sensitive behavior as weak polybase due to the large quantities of amino groups on their chains. Its solubility in aqueous media depends on variables such as pH, temperature and ionic strength of the dissolving medium. Both polymers can be easily dissolved at low pH but are insoluble at higher pH ranges. The mechanism by which these polymers are soluble in aqueous medium involves the protonation of amine groups of the polymers under low pH conditions. This protonation leads
Abbreviations: EuE100, Eudragit E100, Chi, chitosan, CAT, catalase. ∗ Corresponding author. Tel.: +54 341 4804592; fax: +54 341 4804598. E-mail address: [email protected] (B. Farruggia). 0141-8130/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2009.04.009
to chain repulsion, diffusion of proton and counter ions together with water inside the gel and dissociation of secondary interactions. Due to its positively charged amine groups, chitosan and Eudragit E100 interact with negatively charged molecules through coulombic forces [3,4]. If the negatively charged molecules are proteins, non-soluble complexes are formed which can be separated from the other macromolecules by simple precipitation. Production of proteins is a prime biotechnological application which includes upstream and downstream processing steps to obtain the final product in the desired purified form, the downstream processing often being the most expensive one. Bioseparation steps for the recovery of the final product can account for 50–80% of overall production costs. Most purification technologies use precipitation of proteins as one of the initial operations aimed at concentrating the product for further downstream steps [5]. Precipitation is a common approach to obtain enzymes and other macromolecules. This technique offers the possibility of concentrating and purifying the target macromolecule at low cost. Polyelectrolyte precipitation uses a poly-charged macromolecule of opposite electrical charge to the target macromolecule, forming a soluble protein–polyelectrolyte complex under desired experimental conditions; these complexes interact among each other, producing insoluble macroaggregates. This is a suitable method for protein isolation because very low polyelectrolyte concentrations are used (up to 1%, w/v). This method sometimes offers a high selectivity and the insoluble complex can be re-dissolved by a change in pH or by addition of salt to recover the target protein [6].
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In this work we studied the capacity to form complexes between chitosan and Eudragit E100 and a model protein such as bovine liver catalase (pI ≈ 5.5) with the aim of applying this protein precipitation technique as a first step in the isolation of enzymes in scaling up.
Absorbance measurements were recorded on a Jasco 550 spectrophotometer. The sample temperature was controlled by a peltier heating and measured with a thermocouple immersed inside the cuvette. The heating rate was 1 ◦ C/min. The data absorbances vs. temperature were collected by using the software provided by the instrument manufacturer.
2. Materials and methods 2.4. Circular dichroism spectra 2.1. Chemical Liver bovine catalase (CAT), chitosan (Chi) – minimum 75% desacetylation grade, given by the manufacturer – were purchased from Sigma (MO, USA) and used without further purification. Eudragit E100 was gently donated by Etilfarma. Chi was dissolved in acetic acid 0.1 M at a concentration of 2% (w/v). EuE100 was dissolved in clorhidric acid 0.5 M at a concentration of 5% (w/v).
Circular dichroism (CD) scan of a solution of CAT without polymer was carried out and the redisolved polymer-precipitated proteins were also scanned using a Jasco spectropolarimeter, model J-8150. The ellipticity values [] were obtained in millidegrees (mdeg) directly from the instrument. The cell path length of 0.1 cm was used for the spectral range 200–250 nm. In all cases, five scans were made. 2.5. Measurements of the enzymatic activity of CAT
2.2. Proteins turbidimetric titration curves with chitosan and Eudragit E100 The formation of the insoluble polymer–protein complex was followed by turbidimetric titration [6]. Tris–HCl, sodium citrate buffer solutions (10 mL) with a fixed protein concentration were titrated at 25 ◦ C in a glass cell with the polymer solution as the titrant. To avoid changes in pH during titration, both the catalase and the polyelectrolyte solutions were adjusted to the same pH value. The absorbance at 600 nm of solution was used to follow the protein–polyelectrolyte complex formation and plotted vs. the total concentration of polymer in the tube. These plots were fitted with a hyperbolic equation. Solution absorbances were measured using a Jasco 520 spectrophotometer with a thermostatized cell of 1 cm of path length.
Catalase catalyzes the decomposition of hydrogen peroxide into water and oxygen. The catalase enzymatic activity was determined spectrophotometrically by monitoring the decrease in hydrogen peroxide (3 mM) absorption at 240 nm in a buffer phosphate medium 50 mM pH 7.4. In this work 1 unit of catalase activity was defined as the amount of enzyme that catalyzes the reaction of 1 mmol of hydrogen peroxide per minute. 2.6. Quenching of the protein native fluorescence by acrylamide The quenching of the protein tryptophan fluorescent residues was carried out by titration with acrylamide in the presence and absence of Chi. The data were analyzed using the mathematical model for dynamic quenching according to Lakowicz [8]:
2.3. Catalase thermal stability
F0 = 1 + KD [Q] F
Thermally induced unfolding was monitored by absorbance at 280 nm, as it was previously reported [7]. Data were analyzed assuming an approximation of a two-state model of denaturation where only the native and unfolded states were significantly populated and the absorptivity coefficients of both states were different. We used non-linear least squares to fit the absorbance vs. temperature (T) data. The temperature at the mid-point of denaturation (Tm) was determined and the unfolded protein fraction was calculated from
where F0 and F are the protein fluorescence emission at 340 nm, when the protein was excited at 280 nm in the absence and presence of a quencher, respectively; KD is the Stern–Volmer constant related to the lifetime of the fluorophore and the diffusion coefficient of the quencher, and [Q] the quencher concentration.
Absi − AbsN ˛= AbsD − AbsN
(1)
where ˛ is the unfolded protein fraction, AbsN and AbsD are the absorptivities of the native and unfolded states, respectively, Absi is the absorbance at a given temperature. The equilibrium constant for the unfolded process temperature dependence can be calculated as follows: ˛ (2) K= 1−˛ the free energy (G◦ ) was calculated as: G◦ = −RT ln K From a plot of G◦
(3) vs. T, the unfolded S◦
was calculated according
to: ∂G◦ = −S ◦ ∂T
(4)
The enthalpic change ◦
◦
H = G + TS
◦
(H◦ )
was calculated from the equation: (5)
(6)
3. Results 3.1. Chi and EuE100 acid–base titration curves In order to estimate the pKa of polymer amino groups and analyze how the protein presence affects the acid–base polymer state, in the presence and absence of CAT, titration curves were made (see inset in Fig. 1). EuE100 showed typical titration curves with a pKa of 6.7 in the presence or absence of CAT. The Chi was dissolved in 0.1 M HAc, therefore the chitosan acid–base titration curve only showed the titration of acetic acid and did not allow us to study the polymer acid–base behavior. For the same reason, the chitosan acid–base titration curve and the CAT with chitosan acid–base titration curve could be superposed (data not shown). This might indicate that the protein does not modify the polymer acid–base state in this way. 3.2. Solubility curves of Chi and EuE100 vs. the medium pH in the catalase presence It is well known that the solubility of Chi and EuE100 is highly dependent on medium pH, because the protonation state of the amine groups of these polymers induces the repulsion between them. Titration curves of Chi and EuE100 solutions were obtained to
V. Boeris et al. / International Journal of Biological Macromolecules 45 (2009) 103–108
Fig. 1. Absorbance at 600 nm of Chi and EuE100 solution vs. the medium pH. Polymer concentration: 0.1% (w/v), the medium pH was varied adding increasing amount of NaOH or HCl. The CAT concentration was 0.31 mg/mL. Temperature 25 ◦ C. Medium acetate–phosphate 50mM–50 mM. Inset: EuE100 acid–base titration in the presence and absence of CAT.
characterize the polyelectrolytes used in this work as shown in Fig. 1 and expressed as the medium absorbance at 600 nm. The medium pH variation was obtained by adding NaOH or HCl aliquots, allowing the system to reach equilibrium and then the pH of the medium was measured. The curve was also obtained in the presence of CAT in order to find the best protein precipitation range with the flexible chain polymers. A typical sigmoidal titration curve was obtained in both cases. The polymer titration curve in the protein presence cannot overlap the curve in the absence of the protein. CAT has an isoelectrical point of 5.5. From this pH value it has a negative net charge and it would begin to interact with the polymers, which have positives charges by electrostatic attraction. Thus, a complex whose absorbance values are very much larger than the observed values in the case of the polymers alone is formed. These solubility curves allow us to find the pH value in which the complexes are formed and the polymers are soluble. Both complexes CAT–polymers have the maximum precipitation around pH 6. 3.3. Turbidimetric titration of protein with the polymers To assay the capacity of these polymers to interact with CAT, a pH value of 6.0 was selected to carry out the polymer–CAT complex formation from the results obtained from the previous curves. Under this pH value, CAT has a net negative electrical charge. Fig. 2 shows the absorbance dependence at 600 nm when the CAT, at a constant concentration, is titrated with Chi and EuE100, respectively. The protein showed a curve with a hyperbolic shape while the non-precipitated enzymatical activity in the supernatant showed a decrease in the same way as the precipitate was formed, which reflects the disappearance of the macromolecule in the supernatant. However, at high polymer concentrations, the protein precipitation was not complete. The supernatant enzymatic activity curves vs. polymer concentration showed a plateau that indicates that it is not possible to precipitate 100% of the protein in the solution, so there were still proteins remaining in the solution (20% of CAT with both polymers). From the non-linear fitting of the turbidimetry titration curve, the protein–polymer ratio which corresponds to the stoichiometry of insoluble complex formation was calculated as shown in Table 1. As it was pointed out by other report [9], this protein–polyelectrolyte ratio corresponds to the final state of the
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Fig. 2. Titration of CAT 0.31 mg/mL with Chi and EuE100. Medium buffer phosphate 50 mM, pH 6.0. Temperature 25 ◦ C. The enzyme activity in the supernatant vs. polymer total concentration was also determined. (䊉) Turbidity (CAT + Chi); ( ) turbidity (CAT + EuE100); () remaining activity (CAT + Chi); () remaining activity (CAT + EuE100).
complex in its non-soluble form. It is well-demonstrated that the formation of this complex is produced by two steps [10]: first, the formation of a primary protein–polymer complex which is soluble; and second, the soluble complex particles interact among them to form high molecular and non-soluble aggregates which are determined by the turbidimetry titration. The above stoichiometry ratio corresponds to the formation of this last state. Therefore, this value is a mean value of the CAT/polymer masses ratio in the precipitate. The protein–polyelectrolyte ratio values calculated at the plateau of the turbidimetric curves are important because they allow us to calculate the minimal polymer amount to precipitate the protein. High CAT/polymer ratio with values from 1.6 g CAT for gram Chi and 3.4 g CAT for gram EuE100 were obtained, which is consistent with the results reported for the precipitation of other proteins by polyelectrolytes [9]. The mass of the polyelectrolyte was expressed in grams because Chi is a polydisperse polymer since it is a natural product. The necessary quantity of polymers for protein precipitation was lower for EuE100 than for Chi. This fact is very important because EuE100 is a polymer that can be added in low proportion for protein purification due to its potential toxicity but Chi, which is necessary in greater quantities, might be consumed by human beings. However, both polymers are required in a very low concentration value compared with the value of others polyelectrolytes such as polyethyleneimine, polyvinylsulphonate or polyacrylic acid required to precipitate some proteins or the classical protein precipitant such as inorganic cations and anions [9,11,12]. Therefore, Chi and EuE100 precipitation appears to be an excellent method to precipitate slightly acid proteins due to the fact that it is nonexpensive and uses limited amounts of polyelectrolytes. This is important in designing scaling-up methods to precipitate proteins by using a polyelectrolyte because the target proteins are present in high volume of solution so small masses of these polyelectrolytes are necessary to precipitate the desired enzyme completely. Table 1 Mass ratio of the insoluble complexes formed by CAT and Chi or EuE100. System
Stoichiometry (g CAT/g polymer)
CAT - chitosan CAT - Eudragit E100
1.6 ± 0.2 3.4 ± 0.1
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Fig. 3. Turbidimetric titration curve of CAT 0.31 mg/mL by Chi and EuE100 (inset) at increasing ionic strength given by NaCl. Medium condition sodium phosphate buffer 50 mM, pH 6.0. Temperature 25 ◦ C.
3.4. Turbidimetric titration of protein with the polymers in the presence of electrolytes When the precipitation curves were carried out at different NaCl concentrations, they showed were consistent with a net electrostatic mechanism proposed for this complex formation since the increase in the NaCl concentration at level around 0.3 M inhibited the CAT–Chi or CAT–EuE100 complex formation. In the CAT–Chi complex case the NaCl effect is similar with any NaCl concentration but the NaCl effect on the CAT–EuE100 complex is different as it can be seen in Fig. 3. The CAT–Chi complex formation is markedly diminished in the presence of NaCl at any concentration and all the plots are coincident. On the other hand, the CAT–EuE100 complex dissolves gradually in the presence of NaCl. This is possibly due to a different effect of the ionic strength on the polymers. The sodium salt halides (Cl− , Br− and F− ) were assayed on the complex formation. In the presence of halides, the CAT–Chi complex showed superposed plots (data not shown), suggesting that no ordered water is involved in the protein–polymer interaction, so a coulombic mechanism is the one which takes part in this interaction. However, the CAT–EuE100 complex formation diminishes so much as the Hofmeister series increases, as it can be seen in Fig. 4. Both results might indicate that there is a hydrophobic contribution in the formation of the CAT–EuE100 complex because the disordered ions of the structured water affect this process. The presence of a non-electrostatic component in the polyelectrolyte protein complex formation has been reported [13] as well as the presence of a hydrophobic effect where the structured water plays an important role.
Fig. 4. Turbidimetric titration curve of CAT 0.31 mg/mL by EuE100 in the presence of sodium salt halides at constant ionic strength (100 mM).Medium condition: sodium phosphate buffer 50 mM, pH 6.0. Temperature 25 ◦ C.
[14]. From these curves, the middle transition temperature (Tm) was calculated by a non-lineal fitting of the data. Chi and EuE100 do not induce significant differences on the thermodynamic stability of both proteins because no important change in the Tm value was observed. Applying Eqs. (4)–(6), the thermodynamic functions (H◦ and S◦ ) of both proteins were obtained as shown in Table 2. A positive entropic change for the thermal denaturation process was found as well as in other proteins [15]. This change is associated with a loss of ordered water around the hydrophobic moieties of the protein and a change in the tertiary protein structure. However, in the presence of Chi and EuE100, a decrease in the entropic change was observed for CAT, suggesting a slight stabilization of the protein in the complex, in agreement with the protection effect of the polyelectrolytes on the protein against the temperature. CAT unfolding enthalpic change was slightly decreased by the presence of these polycations, suggesting a bond labilization in the complex according to the observed positive entropic change. The values obtained for both functions are balanced and account for the fact that the polymers do not affect significantly the Gibbs
3.5. Thermal stability of protein in the presence of polymer The interaction between a polymer and a protein may induce conformational changes in the protein tertiary and secondary structure, with a loss of its thermodynamical stability and biological activity. The thermal unfolding of CAT was analyzed by measuring the protein absorbance change at 280 nm with increasing temperatures, as it was previously reported [7]. Fig. 5 shows the unfolded fraction vs. the temperature for CAT in the absence and presence of EuE100 and Chi. It can be seen that the presence of polyelectrolyte changes the shape of the curve and there is a decrease in the slope of the middle zone of the curve, which is consistent with a loss of the cooperative effect associated with the unfolding process
Fig. 5. Unfolding thermal shape of CAT (0.31 mg/mL) in the absence and presence of Chi 0.1% (w/v) or EuE100 0.1% (w/v). The data have been expressed as unfolding protein fraction vs. the temperature. Heating rate: 1 ◦ /min. Medium buffer acetate 50 mM, pH 4.0.
V. Boeris et al. / International Journal of Biological Macromolecules 45 (2009) 103–108
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Table 2 Thermodynamic function values associated with the thermal unfolded process of CAT in the absence and presence of Chi and EuE100 calculated at 25 ◦ C. System
Tm (◦ C)
G◦ (kcal/mol)
S◦ (cal/(mol K))
H◦ (kcal/mol)
CAT CAT - chitosan CAT - Eudragit E100
61.3 ± 0.1 62.0 ± 0.5 61.6 ± 0.3
1.9 ± 0.1 1.40 ± 0.09 1.59 ± 0.07
43.5 ± 0.2 31.6 ± 0.4 37.1 ± 0.3
14.9 ± 0.8 10.8 ± 0.9 12.7 ± 0.8
energies involved in the denaturation process so, the equilibrium between native and denaturated form remains unaltered.
Table 3 Dynamic quenching constant values (KD ) for the quenching of the CAT fluorescence with acrylamide.
3.6. Fluorescence and circular dichroism spectra of CAT in the presence of cationic polymers
System
KD (L/mol)
CAT CAT - chitosan CAT - Eudragit E100
3.45 ± 0.05 3.23 ± 0.03 3.03 ± 0.04
Spectroscopy techniques such as native fluorescence emission and circular dichroism are very useful to obtain information about the interaction between a protein and another molecule. Protein CD spectrum gives information about changes induced in the secondary structure of macromolecules through its alpha helix content. We have obtained the circular dichroism spectrum of CAT in the absence and presence of Chi and EuE100. No significant modifications in the CD spectrum of CAT in the presence of Chi or EuE100 are observed as shown in Fig. 6, which suggests that the polymers do not modify the secondary structure of the protein. This finding is important since in the design process of an isolation and purification method by polyelectrolyte precipitation, it is necessary for the polyelectrolyte not to change the enzyme biological activity by loss of its secondary and tertiary structure. Neither Chi nor EuE100 induce any modifications in the native fluorescence spectrum of CAT (data not shown), which suggests that the polymers do not modify its tryptophan environment. However, to obtain more information about the behavior of polymer interaction with the protein surface, the quenching of the native fluorescence of tryptophan of CAT in the presence of Chi and EuE100 was assayed. The native fluorescence quenching of a protein using a quencher, such as acrylamide, allows to obtain information about the ability of the quencher molecule to interact with the tryptophan residues accessible to the solvent. CAT was titrated with acrylamide in the absence and presence of Chi and EuE100; the data were expressed as Stern–Volmer plot. No significant differences between the plot slopes were found for CAT (data not shown), in agreement with the fact that the polycations do not change the solvent accessibility to the quencher. By applying Eq. (6), the quenching constant
Fig. 6. CD spectra of CAT 0.31 mg/mL in the presence and absence of Chi 0.1% (w/v) or EuE100 0.1% (w/v). Medium: buffer acetate 50 mM, pH 4.0. Temperature 25 ◦ C.
(KD ) was calculated. The values are shown in Table 3. No differences in the KD values were observed in the presence of Chi and EuE100, which suggests there are no modifications of the tryptophan residues by the polymer. [7]. 4. Discussion Current methods of protein purification involve an extensive series of steps and processes that increase the cost of the final product. New techniques for large-scale protein separation are, therefore, of interest. One of these involves the addition of polyelectrolytes, leading to selective protein phase separation. Proteins interact strongly with both synthetic and natural polyelectrolytes. These interactions are modulated by such variables as pH and ionic strength, and may result in soluble or insoluble complexes, or the formation of amorphous precipitates. This phase separation results from the electrostatic interactions between the protein and the polyelectrolyte which, at very low ionic strength, results in tight ion-pairing and the formation of amorphous precipitates. There are a lot of synthetic polyelectrolytes, such as EuE100, proposed as protein precipitant and, therefore, as a method to isolate enzymes of interest. However, very few of them are non-toxic and a great part of the polyelectrolyte precipitated together with the target enzyme cannot be removed. Chitosan is one of the few cationic biopolymers in nature. It is biocompatible, biodegradable, nonimmunogenic, and non-toxic in animal tissues. At present the uses of Chi have been increased for design downstream processing methods in the biotechnological processes, due to the fact that it is not expensive and has mild properties for the macromolecules. Some works that have applied the polycations capacity to precipitate proteins by using spectroscopy and viscosimetry measurements, have determined that these polymers forms complexes with different proteins but the microstructure of the protein become affected [16]. However, these authors did not determine the formation mechanism of the insoluble polymer–protein complex. Montilla et al. [17] have studied the beta lactoglobulin–Chi complex formation as a method of isolation and purification of this protein from milk whey, without showing data about the stoichiometry of complex formation. One of the most important finding from the present results is that the polymers used do not modify the secondary and tertiary structure of the assayed proteins as it was determined by the CD spectrum of the dissolved CAT–polymer complexes. This finding is consistent with the result found from the fluorescence emission of native protein. Additionally, these polymers do not affect significatively the CAT thermal stability. These results determined that Chi and EuE100 could be used as a potential framework to be
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applied in the isolation of other macromolecules by a precipitation method. 5. Conclusion Polyelectrolyte precipitation uses very low polymer concentration which makes this technique non-expensive. Also, the solution has low viscosity and is of easy application in scale up. The use of a non-toxic and natural polymer such as chitosan, adds another favourable quality to this methodology. However, it is necessary to develop experimental measurements to determine the influences of experimental variables of the medium on the complex formation and dissolution. In the purification process of CAT would be used EuE100 since the amounts required of this polymer needed to precipitate the same amount of enzyme are lower than the Chi. This fact may allow designing processes where this step affect with minor costs to the global downstream processing. Acknowledgements This work was supported by a grant from FoNCyT PICT0612476/02 and CONICET PIP5053. We thank María Robson, Geraldine Raimundo, Mariana De Sanctis and Marcela Culasso for the language correction of the manuscript.
References [1] J.G. LeHoux, G. Dupuis, Carbohydrate Polymers 68 (2007) 287–294. [2] M. George, T.E. Abraham, Journal of Controlled Release 114 (2006) 1–14. [3] J. Wu, M. Luan, J. Zhao, International Journal of Biological Macromolecules 39 (2006) 185–191. [4] M. Hori, H. Onishi, Y. Machida, International Journal of Pharmaceutics 297 (2005) 223–234. [5] R. Scopes, Protein Purification, Springer Verlag, New York, 1988. [6] T. Matsudo, K. Ogawa, E. Kokufuta, Biomacromolecules 4 (2003) 1794– 1799. [7] V. Boeris, B. Farruggia, B. Nerli, D. Romanini, G. Picó, International Journal of Biological Macromolecules 41 (2007) 286–294. [8] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1983. [9] D. Romanini, M. Braia, R. Giatte Angarten, W. Loh, G. Picó, Journal of Chromatography B 857 (2007) 25–31. [10] E. Seyrek, P.L. Dubin, C. Tribet, E.A. Gamble, Biomacromolecules 4 (2003) 273–282. [11] C. Holler, D. Vaughan, C. Zhang, Journal of Chromatography A 1142 (2007) 98–105. [12] A. Manzur, D. Spelzini, B. Farruggia, D. Romanini, G. Picó, Journal of Chromatography B 860 (2007) 63–68. [13] V. Boeris, D. Spelzini, J. Peleteiro Salgado, G. Picó, D. Romanini, B. Farruggia, Biochimica et Biophysica Acta, General Subjects 1780 (2008) 1032–1037. [14] R.J. Ramprakash, V. Doseeva, A. Galkin, W. Krajewski, L. Muthukumar, S. Pullalarevu, E. Demirkan, O. Herzberg, J. Moult, F.P. Schwarz, Analytical Biochemistry 374 (2008) 221–230. [15] J. Fitter, S. Haber-Pohlmeier, Biochemistry 43 (2004) 9589–9599. [16] L. Chen, L. Tianqing, Journal of Biological Macromolecules 42 (2008) 441. [17] A. Montilla, E. Casal, F.J. Moreno, J. Belloque, A. Olano, N. Corzo, International Dairy Journal 17 (2007) 459–464.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Interaction and complex formation between catalase and cationic polyelectrolytes: Chitosan and Eudragit E100 Valeria Boeris, Diana Romanini, Beatriz Farruggia ∗ , Guillemo Picó Bioseparation Lab. Chemical – Physics Department, Faculty of Biochemical and Pharmaceutical Sciences, CONICET, FonCyT and CIUNR, National University of Rosario, Suipacha 570 (S2002RLK) Rosario, Argentina
a r t i c l e
i n f o
Article history: Received 19 March 2009 Received in revised form 16 April 2009 Accepted 18 April 2009 Available online 3 May 2009 Keywords: Catalase Chitosan Eudragit E100 Polyelectrolyte
a b s t r a c t Interactions between catalase and the cationic polyelectrolytes: chitosan and Eudragit E100 have been investigated owing to their scientific and technological importance. These interactions have been characterized by turbidimetry, circular dichroism and fluorescence spectroscopy. It was found that the catalase conformation does not change significantly during the chain entanglements between the protein and the polyelectrolytes. The effects of pH, ionic strength and anions which modify the water structure were evaluated on the polymer–protein complex formation. A net coulombic interaction force between them was found since the insoluble complex formation decreased after the NaCl addition. Both polymers were found to precipitate around 80% of the protein in solution. No modification of the tertiary and secondary protein structure or the enzymatic activity was observed when the precipitate was dissolved by changing the pH of the medium. Chitosan and Eudragit E100 proved to be a useful framework to isolate catalase or proteins with a slightly acid isoelectrical pH by means of precipitation. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Chitosan is a cationic linear polymer, which is the second most abundant polymer in nature after cellulose [1]. Chitin is the primary structural component of the outer skeletons of crustaceans and it is also found in many other species such as mollusks, insects and fungi. The most commonly obtained form of chitosan is the ␣chitosan from crustacean chitin taken from crab and shrimp shell wastes. Chitosan has a wide variety of commercial and biomedical applications which are related to the physical properties of the biopolymer. Eudragit E100 is a cationic copolymer based on dimethylaminoethyl methacrylate and neutral methacrylic esters whose average molecular mass is 135.0000. It is highly used in pharmaceutical industry for immobilization and drug delivery [2]. Chitosan and Eudargit E100 exhibit a pH-sensitive behavior as weak polybase due to the large quantities of amino groups on their chains. Its solubility in aqueous media depends on variables such as pH, temperature and ionic strength of the dissolving medium. Both polymers can be easily dissolved at low pH but are insoluble at higher pH ranges. The mechanism by which these polymers are soluble in aqueous medium involves the protonation of amine groups of the polymers under low pH conditions. This protonation leads
Abbreviations: EuE100, Eudragit E100, Chi, chitosan, CAT, catalase. ∗ Corresponding author. Tel.: +54 341 4804592; fax: +54 341 4804598. E-mail address: [email protected] (B. Farruggia). 0141-8130/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2009.04.009
to chain repulsion, diffusion of proton and counter ions together with water inside the gel and dissociation of secondary interactions. Due to its positively charged amine groups, chitosan and Eudragit E100 interact with negatively charged molecules through coulombic forces [3,4]. If the negatively charged molecules are proteins, non-soluble complexes are formed which can be separated from the other macromolecules by simple precipitation. Production of proteins is a prime biotechnological application which includes upstream and downstream processing steps to obtain the final product in the desired purified form, the downstream processing often being the most expensive one. Bioseparation steps for the recovery of the final product can account for 50–80% of overall production costs. Most purification technologies use precipitation of proteins as one of the initial operations aimed at concentrating the product for further downstream steps [5]. Precipitation is a common approach to obtain enzymes and other macromolecules. This technique offers the possibility of concentrating and purifying the target macromolecule at low cost. Polyelectrolyte precipitation uses a poly-charged macromolecule of opposite electrical charge to the target macromolecule, forming a soluble protein–polyelectrolyte complex under desired experimental conditions; these complexes interact among each other, producing insoluble macroaggregates. This is a suitable method for protein isolation because very low polyelectrolyte concentrations are used (up to 1%, w/v). This method sometimes offers a high selectivity and the insoluble complex can be re-dissolved by a change in pH or by addition of salt to recover the target protein [6].
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In this work we studied the capacity to form complexes between chitosan and Eudragit E100 and a model protein such as bovine liver catalase (pI ≈ 5.5) with the aim of applying this protein precipitation technique as a first step in the isolation of enzymes in scaling up.
Absorbance measurements were recorded on a Jasco 550 spectrophotometer. The sample temperature was controlled by a peltier heating and measured with a thermocouple immersed inside the cuvette. The heating rate was 1 ◦ C/min. The data absorbances vs. temperature were collected by using the software provided by the instrument manufacturer.
2. Materials and methods 2.4. Circular dichroism spectra 2.1. Chemical Liver bovine catalase (CAT), chitosan (Chi) – minimum 75% desacetylation grade, given by the manufacturer – were purchased from Sigma (MO, USA) and used without further purification. Eudragit E100 was gently donated by Etilfarma. Chi was dissolved in acetic acid 0.1 M at a concentration of 2% (w/v). EuE100 was dissolved in clorhidric acid 0.5 M at a concentration of 5% (w/v).
Circular dichroism (CD) scan of a solution of CAT without polymer was carried out and the redisolved polymer-precipitated proteins were also scanned using a Jasco spectropolarimeter, model J-8150. The ellipticity values [] were obtained in millidegrees (mdeg) directly from the instrument. The cell path length of 0.1 cm was used for the spectral range 200–250 nm. In all cases, five scans were made. 2.5. Measurements of the enzymatic activity of CAT
2.2. Proteins turbidimetric titration curves with chitosan and Eudragit E100 The formation of the insoluble polymer–protein complex was followed by turbidimetric titration [6]. Tris–HCl, sodium citrate buffer solutions (10 mL) with a fixed protein concentration were titrated at 25 ◦ C in a glass cell with the polymer solution as the titrant. To avoid changes in pH during titration, both the catalase and the polyelectrolyte solutions were adjusted to the same pH value. The absorbance at 600 nm of solution was used to follow the protein–polyelectrolyte complex formation and plotted vs. the total concentration of polymer in the tube. These plots were fitted with a hyperbolic equation. Solution absorbances were measured using a Jasco 520 spectrophotometer with a thermostatized cell of 1 cm of path length.
Catalase catalyzes the decomposition of hydrogen peroxide into water and oxygen. The catalase enzymatic activity was determined spectrophotometrically by monitoring the decrease in hydrogen peroxide (3 mM) absorption at 240 nm in a buffer phosphate medium 50 mM pH 7.4. In this work 1 unit of catalase activity was defined as the amount of enzyme that catalyzes the reaction of 1 mmol of hydrogen peroxide per minute. 2.6. Quenching of the protein native fluorescence by acrylamide The quenching of the protein tryptophan fluorescent residues was carried out by titration with acrylamide in the presence and absence of Chi. The data were analyzed using the mathematical model for dynamic quenching according to Lakowicz [8]:
2.3. Catalase thermal stability
F0 = 1 + KD [Q] F
Thermally induced unfolding was monitored by absorbance at 280 nm, as it was previously reported [7]. Data were analyzed assuming an approximation of a two-state model of denaturation where only the native and unfolded states were significantly populated and the absorptivity coefficients of both states were different. We used non-linear least squares to fit the absorbance vs. temperature (T) data. The temperature at the mid-point of denaturation (Tm) was determined and the unfolded protein fraction was calculated from
where F0 and F are the protein fluorescence emission at 340 nm, when the protein was excited at 280 nm in the absence and presence of a quencher, respectively; KD is the Stern–Volmer constant related to the lifetime of the fluorophore and the diffusion coefficient of the quencher, and [Q] the quencher concentration.
Absi − AbsN ˛= AbsD − AbsN
(1)
where ˛ is the unfolded protein fraction, AbsN and AbsD are the absorptivities of the native and unfolded states, respectively, Absi is the absorbance at a given temperature. The equilibrium constant for the unfolded process temperature dependence can be calculated as follows: ˛ (2) K= 1−˛ the free energy (G◦ ) was calculated as: G◦ = −RT ln K From a plot of G◦
(3) vs. T, the unfolded S◦
was calculated according
to: ∂G◦ = −S ◦ ∂T
(4)
The enthalpic change ◦
◦
H = G + TS
◦
(H◦ )
was calculated from the equation: (5)
(6)
3. Results 3.1. Chi and EuE100 acid–base titration curves In order to estimate the pKa of polymer amino groups and analyze how the protein presence affects the acid–base polymer state, in the presence and absence of CAT, titration curves were made (see inset in Fig. 1). EuE100 showed typical titration curves with a pKa of 6.7 in the presence or absence of CAT. The Chi was dissolved in 0.1 M HAc, therefore the chitosan acid–base titration curve only showed the titration of acetic acid and did not allow us to study the polymer acid–base behavior. For the same reason, the chitosan acid–base titration curve and the CAT with chitosan acid–base titration curve could be superposed (data not shown). This might indicate that the protein does not modify the polymer acid–base state in this way. 3.2. Solubility curves of Chi and EuE100 vs. the medium pH in the catalase presence It is well known that the solubility of Chi and EuE100 is highly dependent on medium pH, because the protonation state of the amine groups of these polymers induces the repulsion between them. Titration curves of Chi and EuE100 solutions were obtained to
V. Boeris et al. / International Journal of Biological Macromolecules 45 (2009) 103–108
Fig. 1. Absorbance at 600 nm of Chi and EuE100 solution vs. the medium pH. Polymer concentration: 0.1% (w/v), the medium pH was varied adding increasing amount of NaOH or HCl. The CAT concentration was 0.31 mg/mL. Temperature 25 ◦ C. Medium acetate–phosphate 50mM–50 mM. Inset: EuE100 acid–base titration in the presence and absence of CAT.
characterize the polyelectrolytes used in this work as shown in Fig. 1 and expressed as the medium absorbance at 600 nm. The medium pH variation was obtained by adding NaOH or HCl aliquots, allowing the system to reach equilibrium and then the pH of the medium was measured. The curve was also obtained in the presence of CAT in order to find the best protein precipitation range with the flexible chain polymers. A typical sigmoidal titration curve was obtained in both cases. The polymer titration curve in the protein presence cannot overlap the curve in the absence of the protein. CAT has an isoelectrical point of 5.5. From this pH value it has a negative net charge and it would begin to interact with the polymers, which have positives charges by electrostatic attraction. Thus, a complex whose absorbance values are very much larger than the observed values in the case of the polymers alone is formed. These solubility curves allow us to find the pH value in which the complexes are formed and the polymers are soluble. Both complexes CAT–polymers have the maximum precipitation around pH 6. 3.3. Turbidimetric titration of protein with the polymers To assay the capacity of these polymers to interact with CAT, a pH value of 6.0 was selected to carry out the polymer–CAT complex formation from the results obtained from the previous curves. Under this pH value, CAT has a net negative electrical charge. Fig. 2 shows the absorbance dependence at 600 nm when the CAT, at a constant concentration, is titrated with Chi and EuE100, respectively. The protein showed a curve with a hyperbolic shape while the non-precipitated enzymatical activity in the supernatant showed a decrease in the same way as the precipitate was formed, which reflects the disappearance of the macromolecule in the supernatant. However, at high polymer concentrations, the protein precipitation was not complete. The supernatant enzymatic activity curves vs. polymer concentration showed a plateau that indicates that it is not possible to precipitate 100% of the protein in the solution, so there were still proteins remaining in the solution (20% of CAT with both polymers). From the non-linear fitting of the turbidimetry titration curve, the protein–polymer ratio which corresponds to the stoichiometry of insoluble complex formation was calculated as shown in Table 1. As it was pointed out by other report [9], this protein–polyelectrolyte ratio corresponds to the final state of the
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Fig. 2. Titration of CAT 0.31 mg/mL with Chi and EuE100. Medium buffer phosphate 50 mM, pH 6.0. Temperature 25 ◦ C. The enzyme activity in the supernatant vs. polymer total concentration was also determined. (䊉) Turbidity (CAT + Chi); ( ) turbidity (CAT + EuE100); () remaining activity (CAT + Chi); () remaining activity (CAT + EuE100).
complex in its non-soluble form. It is well-demonstrated that the formation of this complex is produced by two steps [10]: first, the formation of a primary protein–polymer complex which is soluble; and second, the soluble complex particles interact among them to form high molecular and non-soluble aggregates which are determined by the turbidimetry titration. The above stoichiometry ratio corresponds to the formation of this last state. Therefore, this value is a mean value of the CAT/polymer masses ratio in the precipitate. The protein–polyelectrolyte ratio values calculated at the plateau of the turbidimetric curves are important because they allow us to calculate the minimal polymer amount to precipitate the protein. High CAT/polymer ratio with values from 1.6 g CAT for gram Chi and 3.4 g CAT for gram EuE100 were obtained, which is consistent with the results reported for the precipitation of other proteins by polyelectrolytes [9]. The mass of the polyelectrolyte was expressed in grams because Chi is a polydisperse polymer since it is a natural product. The necessary quantity of polymers for protein precipitation was lower for EuE100 than for Chi. This fact is very important because EuE100 is a polymer that can be added in low proportion for protein purification due to its potential toxicity but Chi, which is necessary in greater quantities, might be consumed by human beings. However, both polymers are required in a very low concentration value compared with the value of others polyelectrolytes such as polyethyleneimine, polyvinylsulphonate or polyacrylic acid required to precipitate some proteins or the classical protein precipitant such as inorganic cations and anions [9,11,12]. Therefore, Chi and EuE100 precipitation appears to be an excellent method to precipitate slightly acid proteins due to the fact that it is nonexpensive and uses limited amounts of polyelectrolytes. This is important in designing scaling-up methods to precipitate proteins by using a polyelectrolyte because the target proteins are present in high volume of solution so small masses of these polyelectrolytes are necessary to precipitate the desired enzyme completely. Table 1 Mass ratio of the insoluble complexes formed by CAT and Chi or EuE100. System
Stoichiometry (g CAT/g polymer)
CAT - chitosan CAT - Eudragit E100
1.6 ± 0.2 3.4 ± 0.1
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Fig. 3. Turbidimetric titration curve of CAT 0.31 mg/mL by Chi and EuE100 (inset) at increasing ionic strength given by NaCl. Medium condition sodium phosphate buffer 50 mM, pH 6.0. Temperature 25 ◦ C.
3.4. Turbidimetric titration of protein with the polymers in the presence of electrolytes When the precipitation curves were carried out at different NaCl concentrations, they showed were consistent with a net electrostatic mechanism proposed for this complex formation since the increase in the NaCl concentration at level around 0.3 M inhibited the CAT–Chi or CAT–EuE100 complex formation. In the CAT–Chi complex case the NaCl effect is similar with any NaCl concentration but the NaCl effect on the CAT–EuE100 complex is different as it can be seen in Fig. 3. The CAT–Chi complex formation is markedly diminished in the presence of NaCl at any concentration and all the plots are coincident. On the other hand, the CAT–EuE100 complex dissolves gradually in the presence of NaCl. This is possibly due to a different effect of the ionic strength on the polymers. The sodium salt halides (Cl− , Br− and F− ) were assayed on the complex formation. In the presence of halides, the CAT–Chi complex showed superposed plots (data not shown), suggesting that no ordered water is involved in the protein–polymer interaction, so a coulombic mechanism is the one which takes part in this interaction. However, the CAT–EuE100 complex formation diminishes so much as the Hofmeister series increases, as it can be seen in Fig. 4. Both results might indicate that there is a hydrophobic contribution in the formation of the CAT–EuE100 complex because the disordered ions of the structured water affect this process. The presence of a non-electrostatic component in the polyelectrolyte protein complex formation has been reported [13] as well as the presence of a hydrophobic effect where the structured water plays an important role.
Fig. 4. Turbidimetric titration curve of CAT 0.31 mg/mL by EuE100 in the presence of sodium salt halides at constant ionic strength (100 mM).Medium condition: sodium phosphate buffer 50 mM, pH 6.0. Temperature 25 ◦ C.
[14]. From these curves, the middle transition temperature (Tm) was calculated by a non-lineal fitting of the data. Chi and EuE100 do not induce significant differences on the thermodynamic stability of both proteins because no important change in the Tm value was observed. Applying Eqs. (4)–(6), the thermodynamic functions (H◦ and S◦ ) of both proteins were obtained as shown in Table 2. A positive entropic change for the thermal denaturation process was found as well as in other proteins [15]. This change is associated with a loss of ordered water around the hydrophobic moieties of the protein and a change in the tertiary protein structure. However, in the presence of Chi and EuE100, a decrease in the entropic change was observed for CAT, suggesting a slight stabilization of the protein in the complex, in agreement with the protection effect of the polyelectrolytes on the protein against the temperature. CAT unfolding enthalpic change was slightly decreased by the presence of these polycations, suggesting a bond labilization in the complex according to the observed positive entropic change. The values obtained for both functions are balanced and account for the fact that the polymers do not affect significantly the Gibbs
3.5. Thermal stability of protein in the presence of polymer The interaction between a polymer and a protein may induce conformational changes in the protein tertiary and secondary structure, with a loss of its thermodynamical stability and biological activity. The thermal unfolding of CAT was analyzed by measuring the protein absorbance change at 280 nm with increasing temperatures, as it was previously reported [7]. Fig. 5 shows the unfolded fraction vs. the temperature for CAT in the absence and presence of EuE100 and Chi. It can be seen that the presence of polyelectrolyte changes the shape of the curve and there is a decrease in the slope of the middle zone of the curve, which is consistent with a loss of the cooperative effect associated with the unfolding process
Fig. 5. Unfolding thermal shape of CAT (0.31 mg/mL) in the absence and presence of Chi 0.1% (w/v) or EuE100 0.1% (w/v). The data have been expressed as unfolding protein fraction vs. the temperature. Heating rate: 1 ◦ /min. Medium buffer acetate 50 mM, pH 4.0.
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Table 2 Thermodynamic function values associated with the thermal unfolded process of CAT in the absence and presence of Chi and EuE100 calculated at 25 ◦ C. System
Tm (◦ C)
G◦ (kcal/mol)
S◦ (cal/(mol K))
H◦ (kcal/mol)
CAT CAT - chitosan CAT - Eudragit E100
61.3 ± 0.1 62.0 ± 0.5 61.6 ± 0.3
1.9 ± 0.1 1.40 ± 0.09 1.59 ± 0.07
43.5 ± 0.2 31.6 ± 0.4 37.1 ± 0.3
14.9 ± 0.8 10.8 ± 0.9 12.7 ± 0.8
energies involved in the denaturation process so, the equilibrium between native and denaturated form remains unaltered.
Table 3 Dynamic quenching constant values (KD ) for the quenching of the CAT fluorescence with acrylamide.
3.6. Fluorescence and circular dichroism spectra of CAT in the presence of cationic polymers
System
KD (L/mol)
CAT CAT - chitosan CAT - Eudragit E100
3.45 ± 0.05 3.23 ± 0.03 3.03 ± 0.04
Spectroscopy techniques such as native fluorescence emission and circular dichroism are very useful to obtain information about the interaction between a protein and another molecule. Protein CD spectrum gives information about changes induced in the secondary structure of macromolecules through its alpha helix content. We have obtained the circular dichroism spectrum of CAT in the absence and presence of Chi and EuE100. No significant modifications in the CD spectrum of CAT in the presence of Chi or EuE100 are observed as shown in Fig. 6, which suggests that the polymers do not modify the secondary structure of the protein. This finding is important since in the design process of an isolation and purification method by polyelectrolyte precipitation, it is necessary for the polyelectrolyte not to change the enzyme biological activity by loss of its secondary and tertiary structure. Neither Chi nor EuE100 induce any modifications in the native fluorescence spectrum of CAT (data not shown), which suggests that the polymers do not modify its tryptophan environment. However, to obtain more information about the behavior of polymer interaction with the protein surface, the quenching of the native fluorescence of tryptophan of CAT in the presence of Chi and EuE100 was assayed. The native fluorescence quenching of a protein using a quencher, such as acrylamide, allows to obtain information about the ability of the quencher molecule to interact with the tryptophan residues accessible to the solvent. CAT was titrated with acrylamide in the absence and presence of Chi and EuE100; the data were expressed as Stern–Volmer plot. No significant differences between the plot slopes were found for CAT (data not shown), in agreement with the fact that the polycations do not change the solvent accessibility to the quencher. By applying Eq. (6), the quenching constant
Fig. 6. CD spectra of CAT 0.31 mg/mL in the presence and absence of Chi 0.1% (w/v) or EuE100 0.1% (w/v). Medium: buffer acetate 50 mM, pH 4.0. Temperature 25 ◦ C.
(KD ) was calculated. The values are shown in Table 3. No differences in the KD values were observed in the presence of Chi and EuE100, which suggests there are no modifications of the tryptophan residues by the polymer. [7]. 4. Discussion Current methods of protein purification involve an extensive series of steps and processes that increase the cost of the final product. New techniques for large-scale protein separation are, therefore, of interest. One of these involves the addition of polyelectrolytes, leading to selective protein phase separation. Proteins interact strongly with both synthetic and natural polyelectrolytes. These interactions are modulated by such variables as pH and ionic strength, and may result in soluble or insoluble complexes, or the formation of amorphous precipitates. This phase separation results from the electrostatic interactions between the protein and the polyelectrolyte which, at very low ionic strength, results in tight ion-pairing and the formation of amorphous precipitates. There are a lot of synthetic polyelectrolytes, such as EuE100, proposed as protein precipitant and, therefore, as a method to isolate enzymes of interest. However, very few of them are non-toxic and a great part of the polyelectrolyte precipitated together with the target enzyme cannot be removed. Chitosan is one of the few cationic biopolymers in nature. It is biocompatible, biodegradable, nonimmunogenic, and non-toxic in animal tissues. At present the uses of Chi have been increased for design downstream processing methods in the biotechnological processes, due to the fact that it is not expensive and has mild properties for the macromolecules. Some works that have applied the polycations capacity to precipitate proteins by using spectroscopy and viscosimetry measurements, have determined that these polymers forms complexes with different proteins but the microstructure of the protein become affected [16]. However, these authors did not determine the formation mechanism of the insoluble polymer–protein complex. Montilla et al. [17] have studied the beta lactoglobulin–Chi complex formation as a method of isolation and purification of this protein from milk whey, without showing data about the stoichiometry of complex formation. One of the most important finding from the present results is that the polymers used do not modify the secondary and tertiary structure of the assayed proteins as it was determined by the CD spectrum of the dissolved CAT–polymer complexes. This finding is consistent with the result found from the fluorescence emission of native protein. Additionally, these polymers do not affect significatively the CAT thermal stability. These results determined that Chi and EuE100 could be used as a potential framework to be
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applied in the isolation of other macromolecules by a precipitation method. 5. Conclusion Polyelectrolyte precipitation uses very low polymer concentration which makes this technique non-expensive. Also, the solution has low viscosity and is of easy application in scale up. The use of a non-toxic and natural polymer such as chitosan, adds another favourable quality to this methodology. However, it is necessary to develop experimental measurements to determine the influences of experimental variables of the medium on the complex formation and dissolution. In the purification process of CAT would be used EuE100 since the amounts required of this polymer needed to precipitate the same amount of enzyme are lower than the Chi. This fact may allow designing processes where this step affect with minor costs to the global downstream processing. Acknowledgements This work was supported by a grant from FoNCyT PICT0612476/02 and CONICET PIP5053. We thank María Robson, Geraldine Raimundo, Mariana De Sanctis and Marcela Culasso for the language correction of the manuscript.
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