Lysozyme Separation from Tobacco Extract by Aqueous Two-Phase Extraction

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dc.contributor.authorBalasubramaniam, Deepaen
dc.contributor.committeechairZhang, Chenming Mikeen
dc.contributor.committeememberAgblevor, Foster Aryien
dc.contributor.committeememberVan Cott, Kevin E.en
dc.contributor.committeememberCundiff, John S.en
dc.contributor.departmentBiological Systems Engineeringen
dc.date.accessioned2014-03-14T20:31:55Zen
dc.date.adate2003-03-03en
dc.date.available2014-03-14T20:31:55Zen
dc.date.issued2003-02-05en
dc.date.rdate2004-03-03en
dc.date.sdate2003-02-19en
dc.description.abstractTobacco has long been considered as a host to produce large quantities of high-valued recombinant proteins. However, dealing with large quantities of biomass with a dilute concentration of product is a challenge for down-stream processing. Aqueous two-phase extraction (ATPE) has been used in purifying proteins from various sources. It is a protein-friendly process and can be scaled up easily. ATPE was studied for its applicability to recombinant protein purification from tobacco using egg white lysozyme as the model protein. Separate experiments with polyethyleneglycol(PEG)/salt/tobacco extract, and PEG/salt/lysozyme were carried out to determine the partition behavior of tobacco protein and lysozyme, respectively. Two level fractional factorial designs were used to study the effects of factors such as PEG molecular weight, PEG concentration, the concentration of phase forming salt, sodium chloride concentration, and pH on protein partitioning. The results showed that PEG/sodium sulfate system was most suitable for lysozyme purification. Detailed experiments were conducted by spiking lysozyme into the tobacco extract. The conditions with highest selectivity of lysozyme over native tobacco protein were determined using a response surface design. The purification factor was further improved by decreasing the phase ratio along the tie line corresponding to the phase compositions with the highest selectivity. Under selected conditions the lysozyme yield was predicted to be 87% with a purification factor of 4 and concentration factor of 14. The binodial curve and tie line corresponding to the optimal condition for lysozyme recovery for the PEG 3400/sodium sulfate system were developed. The selectivity at the optimal condition was experimentally determined to be 47 with a lysozyme yield of 79.6 % with a purification factor of 10 and a concentration factor of 20. From this study, ATPE was shown to be suitable for initial protein recovery and partial purification from transgenic tobacco.en
dc.description.degreeMaster of Scienceen
dc.identifier.otheretd-02192003-164258en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-02192003-164258/en
dc.identifier.urihttp://hdl.handle.net/10919/31272en
dc.publisherVirginia Techen
dc.relation.haspartfinal_thesis.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectLysozymeen
dc.subjectTobaccoen
dc.subjectProtein Purificationen
dc.subjectAqueous Two-phase extractionen
dc.titleLysozyme Separation from Tobacco Extract by Aqueous Two-Phase Extractionen
dc.typeThesisen
thesis.degree.disciplineBiological Systems Engineeringen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

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