title: Free flow electrophoresis as a method for the purification of enzymes from E. coli cell extract.
abstract: The application of the four techniques of free flow electrophoresis (zone electrophoresis, isotachophoresis, isoelectric focusing and field step electrophoresis) for the purification of proteins from a complex protein mixture was investigated. For this purpose alpha-amylase (EC 3.2.1.1) from Aspergillus oryzae was added and reisolated from E. coli cell extract. The chosen enzyme and the biological extract are models for many industrial separation problems. In optimized experiments purity, purification factor, yield, throughput and efficiency were calculated. The best results were obtained with field step electrophoresis in combination with zone electrophoresis. High purity (0.82 mg enzyme/mg total protein) and high throughput (111 mL sample/h) were achieved using this technique. Field step electrophoresis gave the best throughput (330 mL sample/h), but low purity (0.63 mg enzyme/mg total protein). This technique can also be used for a simple concentration of the sample. With zone electrophoresis a purity of more than 0.95 mg enzyme/mg total protein was obtained, which was the best of all techniques. However, the enzyme concentration was decreased due to dilution with buffer solution after the separation. Isotachophoresis was the most difficult technique, combined with a relatively low recovery of 31% of the enzyme activity. In a purification scheme, free flow electrophoresis is able to substitute one or even several chromatography steps with a negligible loss of biological activity.
authors.affiliation: Analytical Research and Development, Pharma Division, Sandoz, Basel.
authors.forename: R
authors.initials: R
authors.lastname: Kuhn
authors.forename: H
authors.initials: H
authors.lastname: Wagner
mesh_terms: D001236:Aspergillus oryzae / Q000201:enzymology; D004586:Electrophoresis / Q000379:methods*; D004798:Enzymes / Q000302:isolation & purification*; D004926:Escherichia coli / Q000032:analysis; D007525:Isoelectric Focusing; D000516:alpha-Amylases / Q000302:isolation & purification
chemical_list: D004798:Enzymes; D000516:alpha-Amylases