Preparation of the multienzyme system gramicidin S-synthetase 2 with an aqueous three-phase system.
The distribution of gramicidin S-synthetase activity from disrupted cells suspended in aqueous two- and three-phase systems was investigated. An optimized three-phase system containing 5% dextran, 8% Ficoll, 11% PEG and 6.7% disrupted cells was found to be effective in extracting gramicidin S-synthetase activity. The activity yield achieved was higher in comparison to other preparation methods, and the subsequent purification steps were greatly facilitated. The time needed for the preparation of the labile gramicidin S-synthetase was considerably reduced. The combination of the aqueous phase extraction with chromatographic methods yielded 19 mg gramicidin S-synthetase 2 in essentially pure form from 30 g (wet weight) of cells.
D000591:Amino Acid Isomerases / Q000302:isolation & purification*; D000645:Ammonium Sulfate; D001407:Bacillus / Q000201:enzymology; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D010453:Peptide Synthases / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate; D012997:Solvents
D009097:Multienzyme Complexes; D012997:Solvents; D012967:Sodium Dodecyl Sulfate; D000591:Amino Acid Isomerases; D010453:Peptide Synthases; C020831:gramicidin-S-synthetase 2; D000645:Ammonium Sulfate