ORIGINAL ARTICLES   

Minerva Biotecnologica 2012 September;24(3):91-9

Copyright © 2012 EDIZIONI MINERVA MEDICA

lingua: Inglese

Mechanical cell disruption of Escherichia coli for the release of recombinant green fluorescent protein

Mohd Noor S. S. ¹, Tey B. T. ², Tan W. S. ³, Ling T. C. ⁴, Chan E. S. ², Ramanan R. N. ², Ooi C. W. ²

¹ Institute of Bioscience, Universiti Putra Malaysia, Selangor Darul Ehsan, Malaysia; ² Chemical Engineering, School of Engineering, Monash University, Selangor, Malaysia; ³ Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences University Putra Malaysia, Selangor Darul Ehsan, Malaysia; ⁴ Institute of Biological Sciences, Faculty of Science, University of Malaya, Lembah Pantai, Kuala Lumpur, Malaysia

Aim. Recombinant green fluorescent protein (GFP) is expressed intracellularly in Escherichia coli cells; hence, cell disruption is needed for its release prior to the subsequent purification processes. Two mechanical cell disruption methods, namely ultrasonication and bead milling, were employed to release intracellular GFP from Escherichia coli and the performances of both cell disruption methods were studied.
Methods. The effects of biomass concentration, bead loading, and impeller tip speed on the performance of bead milling were investigated. For ultrasonication, the effects of acoustic power, biomass concentration and suspension volume on its performance were examined.
Results. The optimum operating conditions for bead milling were found to be at 70% (v/v) of bead loading, 10% (w/v) of biomass concentration and impeller tip speed of 10 m/s. For ultrasonication, highest GFP release was achieved at acoustic power 70 W, 10% (w/v) of biomass concentration and 20 ml of suspension volume. The maximum GFP release obtained from bead milling and ultrasonication were 6.912 mg/g cell and 13.542 mg/g cell, respectively.
Conclusion. The maximum GFP obtained in ultrasonication was about two times higher than that in the bead milling method. It can be concluded that ultrasonication is a better method in term of product yield, but bead milling is preferable due to its lower cost operational and its ability to process samples in large volume.