Efficient two-step chromatographic purification of penicillin acylase from clarified Escherichia coli ultrasonic homogenate |
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| Institution: | 1. JMI Laboratories, North Liberty, Iowa, USA;2. University of Iowa, Iowa City, Iowa, USA;1. Department of Biology, University of Florence, Via Madonna del Piano 6, I-50019 Sesto F.no, Florence, Italy;2. CRA-ABP Consiglio per la ricerca in agricoltura e l''analisi dell''economia agraria, Centro di ricerca per l''Agrobiologia e la Pedologia, Piazza M. D''Azeglio 30, 50121 Firenze, FI, Italy;3. Department of Paediatric Medicine, Anna Meyer Children''s University Hospital, Viale G. Pieraccini, 24, 50141 Florence, Italy;4. Ghent University, Laboratory of Bacteriology Research, De Pintelaan 185 BA-3/4, 9000 Ghent, Belgium |
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| Abstract: | 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. |
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