ABSTRACT
A new and efficient safe system for the purification of the penicillin acylase from Escherichia coli G271 is presented. It was found that after a selective precipitation with ammnonium sulphate, followed by two chromatographic steps (anion exchange followed by adsorption on hydroxyapatite support), the enzyme was enriched 98 times with a 100% activity recovery. An original way has also been used to study the chromatographic separation of the protein mixture in three major categories on DEAE resin, by an analysis of the concentrations of the different species in the breakthrough curve obtained from a complete saturation of the column.
Subject(s)
Chromatography, Ion Exchange/methods , Escherichia coli/enzymology , Penicillin Amidase/isolation & purification , Absorption , Ammonium Sulfate/chemistry , Chromatography, Gel , DEAE-Cellulose/chemistry , Electrophoresis, Polyacrylamide Gel , Hydroxyapatites/chemistry , Molecular Weight , Penicillin Amidase/analysisABSTRACT
Thermal parametric pumping was experimentally investigated for the concentration and separation of amino acids. Previous theories of parametric pumping were improved by taking into account dissociation equilibria in the liquid phase. Experiments were carried out with a mixture of glutamic acid, aspartic acid, serine and threonine in a highly acidic solution (HCl). A multi-component equilibrium model was mainly used to simulate the experimental results and to investigate the effect of chloride concentration over a wide range. It is shown that it is always possible to concentrate the amino acids and to separate some of them under certain conditions.
Subject(s)
Amino Acids/isolation & purification , Ion Exchange Resins , Chlorides/chemistry , Models, Chemical , Osmolar Concentration , TemperatureABSTRACT
Several techniques for protein extraction were tested for recovering penicillin acylase from a recombinant strain of Escherichia coli. These techniques include chemical [guanidine hydrochloride, Triton X-100, ethylenediaminetetraacetic acid (EDTA), ethanol/toluene], physical (sonication, freeze-and-thawing), and enzymatic (lysozyme) treatments. Best results were obtained with the combined use of guanidine and EDTA. This extraction procedure was optimized, and it was found that 95% of the enzyme was extracted after a 10 m/M EDTA plus 10 mM guanidine treatment at room temperature for 10 h. The purification factor was 25 when compared to disruption by sonication. This extraction method could avoid purification steps for particular applications.
