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Haloferax mediterraneiextracellular, protease, serine-proteases. KCl and other salts, including ammonium salts, were much less efficient The specific requirement of NaCl for enzyme activity and stability Fig. Elena Irma Villarreal Moguel, and Dr. 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.
Stability of extracellular protease activity in different chemical conditions.
¿Qué es la HPLC y Cómo Funciona?
The optical density of the relative enzyme activity was found as above. Such behavior was similar to that of another Hb. Hence, reported purification methods in those cases hidrofobifa included: We may ascribe that failure to inappropriate experimental conditions to preserve enzyme activity. Haloferax mediterraneiinteracciin extracelular, serin-proteasa. In addition, the Hf. Table 3 describes the effect of different ions on the activity of the purified extracellular protease of Hf.
Although some halophilic enzymes may be reactivated from the salt-free solutions [21, ], and this facilitates their purification ed standard methods, i. The above experiments provided important information about the conditions required for the proper handling of an active extracellular enzyme produced by Hf. 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 , sulfated polysaccharides , and cell wall components , of industrial and biomedical interest.
Biochemistry16 Experientia, 29 The effect of NaCl concentration on enzyme stability at high temperature was measured as follows: After washing the column with 2 volumes of starting buffer, a decreasing linear gradient of NaCl from 2. As with most halophilic enzymes , the purified protease of Hf. Previous reports on the purification of halophilic enzymes from Hf.
These features made the application of the above purification techniques inadequate and, therefore, an alternative procedure had to be explored.
Cromatografía de afinidade
The maximum recovered activity assayed at 2. Halobacteria as producers of polyhydroxyalkanoates. Hydrophobic Interaction Chromatography, HIC, has been seldom interccion 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 their hydrophobicity .
General and Applied aspects of Halophilic interqccion. In our study we found that exposure of the culture supernatant of Hf. After dialysis, the remaining protease activity was measured as above using a 1: Materials and methods All chemicals and reagents were analytical grade Sigma, Chem. Mediterranei to different kinds of salts, including ammonium sulfate, resulted in a considerable loss of activity.
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]. Unfortunately, they were not successful in the isolation and purification of a hisrofobica enzyme from Hf.
hidrofobia 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.
Optimum salt concentration, pH, and temperature studies on enzyme activity of the purified protease.
All steps were done at room temperature as follows: Hence, the traditional salting-out protein purification and concentration procedure cannot be applied in this case. Saturated solutions of sucrose, and betaine, and 5. The purified extracellular protease activity of Hf. Properties of the purified protease Table 3 describes the effect of different ions on the activity of the purified extracellular protease of 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.
Electrophoresis was done at a constant voltage V until the tracking dye bromophenol blue reached 1 cm from the bottom of the gel. Biopolymer production by Haloferax mediterranei. Once established, we were able to introduce the following method for its isolation and purification: Hence, the following experiments were run with 0.
In order to determine the appropriate experimental conditions for purification of Hf.
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Discussion Enzymes from halophilic archaebacteria are highly unstable at low salt concentrations of neutral salts . The capability of Hf. Also the concentration of ammonium ions affected cell growth and the extracellular enzyme activity of Hf. Modification of protease activity in this case, evidenced the influence of the anion species in enzyme activity. Study of proteases of marine bacteria: Physiological and Biochemical Adaptation in Halophilic Microorganisms. In our case, the extra-cellular protease stability and activity of Hf.
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.