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Ivo Safarik as substrate, the cromatograifa salt NaCl concentration for enzyme activity was determined varying the amount of NaCl in the assay mixture 0 to 4.

¿Qué es la HPLC y Cómo Funciona?

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. All steps were done at room temperature as follows: Haloferax mediterraneiextracellular, protease, serine-proteases. 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.

Elena Irma Villarreal Moguel, and Dr. Optimum sodium chloride concentration for stability of CS proteolytic activity. Table 1 shows the effect of different cations on Hf.

Thus, it may be concluded that se most important cations for Hf. Table 3 describes the effect of different ions on the activity of the purified extracellular protease of Hf. For example, without sodium ions, a complete loss of enzyme activity was noticed Table 2. After incubation, the NaCl concentration was adjusted to 2. Extremophiles, 5, We may ascribe that failure to inappropriate experimental conditions to preserve enzyme activity.


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In order to determine the appropriate experimental conditions for purification of Hf. General and Applied aspects of Halophilic microorganisms. The remaining CS was concentrated about 20 times in an Amicon apparatus Mod.

The purified extracellular protease activity of Hf. Optimum salt concentration, pH, and temperature studies on enzyme activity of the purified protease 1.

The combined effect of such parameters is presented in Figs. 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. The maximum recovered activity assayed at 2.

Cromatografía de afinidade

Biochemistry16 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. Effect of cations on the enzyme production and activity.

Stability of extracellular protease activity in different chemical conditions In order to determine the appropriate experimental conditions for purification of Hf. Biochemistry15 This denaturation is irreversible, as observed also with other halophilic archaebacteria [36, 38, 39]. 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 study we found that exposure of the culture supernatant of Hf. Once established, we were able to introduce the following method for its isolation and purification: We all enjoyed his teachings, friendship, and continuous support through all these years.


The maximum activity for purified Hf. 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.

Biopolymer production by Haloferax mediterranei. Most reports of halophilic enzyme isolation and purification dealt well with enzyme stability in different salts like sodium chloride, potassium chloride, and ammonium sulfate.

Biochemistry17 Study of proteases of marine bacteria: As with most halophilic enzymes, the extracellular proteolytic activity of Hf. 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.

The above experiments provided important information about the conditions required for the proper handling of an active extracellular enzyme produced by Hf. The samples were finally dialyzed against To determine the optimum NaCl concentration required for CS protease stability, two different experiments were carried out: First, for the removal of the exopolysaccharide component a selective ultrafiltration procedure cromatograifa carried out.