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Cromatografía de afinidade

Haloferax mediterraneiextracellular, protease, serine-proteases. Hence, reported purification methods in those cases have included: We all enjoyed his teachings, friendship, and continuous support through all these years.

Academic Press, Orlando,8 To determine the type of salt required for protease stability, samples of 1 mL of CS10, previously diluted 1: 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. Stability of extracellular protease activity in different chemical conditions In order to determine the appropriate experimental conditions for purification of Hf.

A decreasing linear gradient of NaCl from 5. In our study we found that exposure of the culture supernatant 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.

In our case, the extra-cellular protease stability and activity of Hf.

Haloferax mediterraneiproteasa extracelular, serin-proteasa. The effect hidrofoobica salt concentration on both substrate and enzyme may favor their association and therefore help the enzyme function better. Elena Irma Villarreal Moguel, and Dr. Biochemistry16 The samples were finally dialyzed against After 10 min, the solutions were quickly chilled in an ice bath. The temperature and salt concentration effect on enzyme activity was determined as follows: The procedure consisted in homogenizing 20 mg of the insoluble substrate in 2 mL of Tris-HCl 0.


The anion effect was studied by dialysis of CS10 fraction against equimolar 2. In addition, the Hf. To date, several enzymes from Hf.

Exposure to lower or higher salt concentration reduced enzyme activity. Mediterranei to different kinds of salts, including ammonium sulfate, resulted in a considerable loss of activity. The optical density of the relative enzyme activity was found as above.

Acta, The purified extracellular protease activity of Hf. The combined effect of such parameters is presented in Figs. We also found a great loss of activity when the culture supernatant CSEP was kept at room temperature, and that the protease activity yield could be enhanced if the purification process was carried at low temperature; presumably, due to a reduced autohydrolysis.

This denaturation is irreversible, as observed also with other halophilic archaebacteria [36, 38, 39].

Additionally, a negative control was prepared intraccion Tris buffer instead of the culture supernatant. The maximum optical density after 4 days of incubation was 1. Enzymes from halophilic archaebacteria are highly unstable at low salt concentrations of neutral salts [45].

However, with casein Hf.

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Stability of extracellular protease activity in different chemical conditions. The effect of NaCl concentration on Hf.

These features made the application of the above purification techniques inadequate and, therefore, an alternative procedure had to be explored. The above experiments provided important information about the conditions required for the proper handling of an active extracellular enzyme produced by Hf. Accordingly, and based on previous reports [], we studied first the hydrolytic enzyme properties of the crude culture supernatant under intersccion conditions to identify an optimum procedure for its purification and, afterwards, the properties of the active purified enzyme.


Effect of salt concentration on CS proteolytic activity. The reaction was ended removing the insoluble material by filtration through a Whatman filter paper and the absorbance at nm measured against the corresponding blank. Also the concentration of ammonium ions affected cell growth and the extracellular enzyme activity of Hf. As with most halophilic enzymes, the extracellular proteolytic activity of Hf. To determine the optimum NaCl concentration required for CS protease stability, two different experiments were carried out: Hence, the following experiments were run with 0.

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The active fractions that separately emerged from the column Fig. The exopolysaccharide for example, may play an important role in controlling water activity, which can be also modified by the presence of salts according to the nature of the ions involved.

Recibido el 16 de enero del This treatment evidenced the enzyme instability at low salt concentration and led us itneraccion consider the addition of salt to preserve activity whenever required. Previous reports on the purification of halophilic enzymes from Hf. After incubation, the NaCl concentration cromatografis adjusted to 2.