Details
| Original language | English |
|---|---|
| Pages (from-to) | 359-364 |
| Number of pages | 6 |
| Journal | Journal of membrane science |
| Volume | 444 |
| Publication status | Published - 1 Jun 2013 |
Abstract
This study describes the purification of penicillin G amidase (PGA) by ion exchange membrane adsorbers in a one-step-process. Preliminary experiments with high-throughput screening devices in microliter scale (8-strip modules) were performed to find suitable purification strategy and appropriate ion exchange ligands as well as basic process conditions for binding and elution. Best purification results were achieved by strong cation-exchange (S-) membrane adsorbers loaded with 2ml/min enzyme solution at pH 6.0 and eluted at pH 6.0 with 0.05M NaCl, which led to a high yield of bound PGA (98%) without any visible remains of host cell proteins and with a residual enzyme activity of 80-85%. The binding of PGA to the S-membrane was further investigated in an upscaling to milliliter scale with LP15 modules and breakthrough curves were determined by varying the flow rates: the PGA-binding to S-membrane adsorbers is independent of the flow rate. Dynamic binding capacities were estimated to be 0.69mg PGA/cm2 (25.5mg/ml) for 10% breakthrough respectively 0.95mg/cm2 (35.2mg/ml) for 100% breakthrough. Finally, real cell lysate samples from Escherichia coli culture containing PGA were processed under the found optimal conditions. Despite exceeded loading PGA was isolated from this complex mixture successfully fourfold concentrated and with a purification factor of 101.3 and a resulting specific activity of 4.97U/mg.
Keywords
- Ion-exchange chromatography, Membrane adsorber, Penicillin G amidase
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Materials Science(all)
- Chemistry(all)
- Physical and Theoretical Chemistry
- Chemical Engineering(all)
- Filtration and Separation
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In: Journal of membrane science, Vol. 444, 01.06.2013, p. 359-364.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - One-step-purification of penicillin G amidase from cell lysate using ion-exchange membrane adsorbers
AU - Mönster, Andrea
AU - Villain, Louis
AU - Scheper, Thomas
AU - Beutel, Sascha
N1 - Funding information: This research project was supported partly by the German Federal Ministery of Education and Research (Bundesministerium für Bildung und Forschung (BMBF)) within the BIOCATALYSIS2021-program and funded also partly by the Deutsche Forschungsgemeinschaft (DFG) within the REBIRTH cluster of excellence.
PY - 2013/6/1
Y1 - 2013/6/1
N2 - This study describes the purification of penicillin G amidase (PGA) by ion exchange membrane adsorbers in a one-step-process. Preliminary experiments with high-throughput screening devices in microliter scale (8-strip modules) were performed to find suitable purification strategy and appropriate ion exchange ligands as well as basic process conditions for binding and elution. Best purification results were achieved by strong cation-exchange (S-) membrane adsorbers loaded with 2ml/min enzyme solution at pH 6.0 and eluted at pH 6.0 with 0.05M NaCl, which led to a high yield of bound PGA (98%) without any visible remains of host cell proteins and with a residual enzyme activity of 80-85%. The binding of PGA to the S-membrane was further investigated in an upscaling to milliliter scale with LP15 modules and breakthrough curves were determined by varying the flow rates: the PGA-binding to S-membrane adsorbers is independent of the flow rate. Dynamic binding capacities were estimated to be 0.69mg PGA/cm2 (25.5mg/ml) for 10% breakthrough respectively 0.95mg/cm2 (35.2mg/ml) for 100% breakthrough. Finally, real cell lysate samples from Escherichia coli culture containing PGA were processed under the found optimal conditions. Despite exceeded loading PGA was isolated from this complex mixture successfully fourfold concentrated and with a purification factor of 101.3 and a resulting specific activity of 4.97U/mg.
AB - This study describes the purification of penicillin G amidase (PGA) by ion exchange membrane adsorbers in a one-step-process. Preliminary experiments with high-throughput screening devices in microliter scale (8-strip modules) were performed to find suitable purification strategy and appropriate ion exchange ligands as well as basic process conditions for binding and elution. Best purification results were achieved by strong cation-exchange (S-) membrane adsorbers loaded with 2ml/min enzyme solution at pH 6.0 and eluted at pH 6.0 with 0.05M NaCl, which led to a high yield of bound PGA (98%) without any visible remains of host cell proteins and with a residual enzyme activity of 80-85%. The binding of PGA to the S-membrane was further investigated in an upscaling to milliliter scale with LP15 modules and breakthrough curves were determined by varying the flow rates: the PGA-binding to S-membrane adsorbers is independent of the flow rate. Dynamic binding capacities were estimated to be 0.69mg PGA/cm2 (25.5mg/ml) for 10% breakthrough respectively 0.95mg/cm2 (35.2mg/ml) for 100% breakthrough. Finally, real cell lysate samples from Escherichia coli culture containing PGA were processed under the found optimal conditions. Despite exceeded loading PGA was isolated from this complex mixture successfully fourfold concentrated and with a purification factor of 101.3 and a resulting specific activity of 4.97U/mg.
KW - Ion-exchange chromatography
KW - Membrane adsorber
KW - Penicillin G amidase
UR - http://www.scopus.com/inward/record.url?scp=84879452862&partnerID=8YFLogxK
U2 - 10.1016/j.memsci.2013.05.054
DO - 10.1016/j.memsci.2013.05.054
M3 - Article
AN - SCOPUS:84879452862
VL - 444
SP - 359
EP - 364
JO - Journal of membrane science
JF - Journal of membrane science
SN - 0376-7388
ER -