Efficient two-step chromatographic purification of penicillin acylase from clarified Escherichia coli ultrasonic homogenate.
A two-step chromatographic purification procedure from clarified Escherichia coli ultrasonic homogenate was evaluated. The capture step included immobilized metal affinity chromatography with Cu2+ as metal ion. Two elution methods were performed: 1 M NH4Cl and 0.01 M imidazole. Respectively, we obtained a different purification fold (16.5 to 3.15) and a similar result for the recovery of activity (90-99%). The best elution method was chosen for the procedure. The second step, hydrophobic interaction chromatography, gave a 3.8-fold purification with 77.7% of activity. The total procedure gave a 66-fold purification in relation to the initial crude extract with 70% for the recovery of activity and was performed without any conditioning step and at the same pH value.
Sanchez
J
J
Ecole Supérieure de Technologie des Biomolécules de Bordeaux, CNRS-UMR 5544, Université Victor Segalen Bordeaux 2, France.
Verdoni
N
N
Fitton
V
V
Santarelli
X
X
D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D010399:Penicillin Amidase / Q000302:isolation & purification*; D014465:Ultrasonics
D010399:Penicillin Amidase