Isolation of a recombinant intracellular beta-galactosidase by ammonium sulfate fractionation of cell homogenates.
The Escherichia coli beta-galactosidase (EC 3.2.1.23) expressed intracellularly as soluble, biologically active enzyme in the yeast Saccharomyces cerevisiae was recovered from clarified homogenates of the yeast cells by precipitation with crystalline ammonium sulfate. Effects of salt saturation (0-80%) of the homogenate, the initial total protein level (2-20 g.L-1) and the processing pH (6-8) on the enzyme and protein recovery were investigated. As the ammonium sulfate concentration increased, the enzyme was precipitated preferentially and at 30% salt saturation nearly all had been recovered in the precipitate. In contrast, at this salt concentration only 25% of the total protein had precipitated. Preferential precipitation of beta-galactosidase was associated with the hydrophobic nature of this large protein. Complete precipitation of the total protein required salt concentrations exceeding 70% of saturation. The salt concentration needed for complete recovery of the enzyme was not sensitive to the processing pH. Over the salt saturation level of 20-50%, the enzyme precipitation followed the Cohn equation. The salting-out constant was strongly affected by the initial protein level in the homogenate; higher values were observed at lower protein concentrations. The salting-out constant was unaffected by the processing pH; however, the Cohn parameter B was pH dependent in addition to being affected by the initial protein concentration. Within the beta-galactosidase stability range of pH 6-8, pH variations alone (no added salt) proved ineffective in precipitating the enzyme.
Zhang
Z
Z
Department of Chemical Engineering, University of Waterloo, Ontario, Canada.
Chisti
Y
Y
Moo-Young
M
M
D000645:Ammonium Sulfate / Q000737:chemistry*; D005591:Chemical Fractionation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D011506:Proteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase; D000645:Ammonium Sulfate