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cromatografía de líquidos interacción hidrófila · cromatografía de interacción hidrofóbica · cromatografía de intercambio de iones · cromatografía de líquidos. La enzima extracelular, purificada mediante ultra-filtración y Cromatografía de Interacción Hidrofóbica, consiste en una cadena de polipéptido de PM 25, Da. METODO PARA AISLAR Y PURIFICAR CONJUGADOS DE TOXINAS USANDO CROMATOGRAFIA DE INTERACCION HIDROFOBICA. LAS MEZCLAS.

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Comparative Biochemistry and Physiology. Recibido el 16 de enero del Aceptado el 27 de mayo del This enzyme belongs to the serine-protease class and could be useful in a variety of industrial applications.

Haloferax mediterraneiextracellular, protease, serine-proteases. Esta enzima pertenece a la clase de serina-proteasas que pueden encontrar atractivas aplicaciones industriales. Haloferax mediterraneiproteasa extracelular, serin-proteasa. The halophilic microorganisms, represented by the halobacteria extremely halophilic aerobic Archaeabacteriathe moderate halophiles bacteria and some methanogensand several eukaryotic algae, and their products, have been a research subject in modern biotechnology [1].

The halobacteria in particular, which are organisms usually found in habitats where the salt concentration is higher than sea water [], seem to be able to produce a number of metabolites and polymers such as polyhydroxybutirate PHB [5], sulfated polysaccharides [6], and cell wall components [7], of industrial and biomedical interest.

To date, several enzymes from Hf. As with most halophilic enzymes, the extracellular proteolytic activity of Hf. Being aware of the advantages offered by Hydrophobic Interaction Chromatography, a technique that allows the use of a high salt concentration to favor a selective adsorption of the protein on the basis of its hydrophobicity [35], we decided to explore its application in the isolation of the extracellular enzyme responsible of the proteolytic activity shown by Hf.

Accordingly, and based on previous reports [], we studied first the hydrolytic enzyme properties of the crude culture supernatant under different conditions to identify an optimum procedure for its purification and, afterwards, the properties of the active purified enzyme.

All chemicals and reagents were analytical grade Sigma, Chem. Unless indicated otherwise, all solutions and culture media were prepared in 0. Organism and culture conditions. Additionally, a negative control was prepared using Tris buffer instead of the culture supernatant.

Accordingly, one enzyme unit is defined as the amount required to cause an increase in absorption of 0. Effect of salt concentration on CS proteolytic activity. The remaining CS was concentrated about 20 times in an Amicon apparatus Mod. The new concentrated material was designated as CS One mL of CS10 was diluted 1: Cation effect on CS proteolytic activity. After dialysis, the remaining protease activity was measured as above using a 1: Optimum sodium chloride concentration for stability of CS proteolytic activity.

Cromatografía de afinidade

To determine the optimum NaCl concentration required for CS protease stability, two different experiments were carried out: The dialyzed samples were cromatografiw 1: After this period, the proteolytic activity was found using 0. Specific ion df for CS proteolytic activity.

To determine the type of salt required for protease stability, samples of 1 mL of CS10, previously diluted 1: The samples were finally dialyzed against A sample of 0. After washing the column with 2 volumes of starting buffer, a decreasing linear gradient of NaCl from 2.


One mL of CS10 diluted 1: A decreasing linear gradient of NaCl from 5. All steps were done at room temperature as follows: A fresh culture supernatant was subjected to ultrafiltration in an Amicon system using a YM diaflomembrane to separate the high molecular weight exopolysaccharide [41, 42]. This concentrated protease material free of exopolysaccharides was designated as CS-EP. Fractions of 6 mL were collected in tubes containing 5 mL of 5 M NaCl and assayed for protein [43] and protease activity.

Electrophoresis was done at a constant voltage V until the tracking dye bromophenol blue reached 1 cm from the bottom of the gel.

Optimum salt concentration, pH, and temperature studies on enzyme activity of the purified protease. Ivo Safarik as substrate, the optimum salt NaCl concentration for enzyme activity was determined varying the amount of NaCl in the assay mixture 0 to 4. The procedure consisted in homogenizing 20 mg of the insoluble substrate in 2 mL of Tris-HCl 0.

The reaction was ended removing the insoluble material by filtration through a Whatman filter paper and the absorbance interacfion nm measured against the corresponding blank. The enzyme stability at different NaCl concentrations was determined as follows: After incubation, the NaCl concentration was adjusted to 2.

The optical density of the relative enzyme activity was found as above. The enzyme activity was then determined as above. The effect of temperature on enzyme activity was determined as follows: After 10 min, the solutions were quickly chilled in an ice bath. The effect of NaCl concentration on enzyme stability at high temperature was measured as follows: The NaCl concentration hidrofobics adjusted to 2.

The temperature and salt concentration effect on enzyme activity was determined as follows: The activity was determined as above.

PURIFICACIÓN by Nicole Elenter on Prezi

The maximum optical density after 4 days of incubation was 1. Also the concentration of ammonium ions affected cell growth and the extracellular enzyme activity of Hf. Hence, the following experiments were run with 0.

Ion-specificity requirements for Hf. Table 1 shows the effect of different cations on Hf. Cromaotgrafia of extracellular protease activity in different chemical conditions. In order to determine the appropriate experimental conditions for purification of Hf.

This treatment evidenced the enzyme instability at low salt concentration and led us to consider the addition of salt to preserve activity whenever required. For example, without sodium ions, a complete loss of enzyme activity was noticed Table 2. Thus, it may be concluded that the most hdrofobica cations for Hf. The effect of NaCl concentration on Hf.

The maximum recovered activity assayed at 2. Exposure to lower or higher salt concentration reduced enzyme activity. The anion effect was studied by dialysis of CS10 fraction against equimolar 2. Modification of protease activity in this case, evidenced interxccion influence of the anion species in enzyme activity.

Saturated solutions of sucrose, and betaine, and 5. It should be borne on lnteraccion that Hf. The exopolysaccharide for example, may play an important role in controlling water activity, which can be also modified by the presence of cromqtografia according hidrofobcia the nature of the ions involved. Crokatografia above interaccioh provided important information about the conditions required for the proper handling of an active extracellular enzyme produced by Hf.

Once established, we were able to introduce the following method for its isolation and purification: First, for the removal of the exopolysaccharide component a selective ultrafiltration procedure was carried out. Then, taking into account the nature extracellularand composition high salt concentration culture medium of the CSEP preparation, Hydrophobic Interaction Chromatography, HIC, was considered the method of choice for the enzyme purification as shown in the following section.


The major peaks, containing also most of the proteolytic activity, were pooled after the corresponding electophoretic analysis revealed no differences in protein pattern between the fractions. A large improvement in enzyme recovery was achieved when the chromatography was carried out in a column equilibrated with 2. The active fractions that separately emerged from the column Fig. Because the employed electrophoresis conditions are known to promote protein disgregation, and because only one protein band in the gel was observed, we have concluded that the extracellular protease of Hf.

Hidrofpbica 3 describes the effect of hidrofobixa ions on the activity of the purified extracellular protease of Hf. The combined effect of such parameters is presented in Figs. All these tests provided some useful information about the properties of the enzyme that led us to conclude that the extracellular enzyme activity of Hf.

Enzymes from halophilic archaebacteria are highly unstable at low salt concentrations of neutral salts [45]. This characteristic imposes many restrictions in their detection and in the choice of an appropriate purification technique. Although some halophilic enzymes may be reactivated from the salt-free solutions [21, ], and this facilitates their purification using standard methods, i.

¿Qué es la HPLC y Cómo Funciona?

Previous reports on the purification of halophilic enzymes cromatografja Hf. In our case, the extra-cellular protease stability and activity of Hf. Most reports of halophilic enzyme isolation and purification dealt well with enzyme stability in different salts like sodium chloride, potassium chloride, and ammonium sulfate.

Hence, reported purification methods in those cases have included: Our study, however, has shown that the extracellular protease interaccikn of Hf. These features made the application of the above purification techniques inadequate and, therefore, an alternative procedure had to be explored.

Hydrophobic Interaction Chromatography, HIC, has been seldom applied in the purification of halophilic enzymes [21, 39, 40, 49], in spite of its inherent advantage of combining gel hydrophobicity and salt concentration for the adsorption-elution phenomenon, that helps to resolve the separation of biomolecules on the basis of hidrovobica hydrophobicity [35].

The technique has been successfully applied, for example, by Kamekura and Seno [39] who were able to purify an extracellular protease from an unidentified strain of a halophilic archaebacterium on Butyl Toyopearl and Phenyl Sepharose. Unfortunately, they were not successful in the isolation and purification of a similar enzyme from Hf.

We may ascribe that failure to inappropriate experimental conditions to preserve enzyme activity. In our study we found that exposure of the culture supernatant of Hf. Mediterranei to different kinds of salts, including ammonium sulfate, resulted in a considerable loss of activity. Hence, the traditional salting-out protein purification and concentration procedure cannot be applied in this case.