{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/31272"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/31272","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Lysozyme Separation from Tobacco Extract by Aqueous Two-Phase Extraction","abstract":"Tobacco 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.","abstract_html":"Tobacco 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.","abstract_has_math":false,"creators":["Balasubramaniam, Deepa"],"institution":"Virginia Tech","degree_name":"Master of Science","degree_level":"masters","degree_discipline":"Biological Systems Engineering","degree_department":"Biological Systems Engineering","school":null,"contributors":[],"advisors":[],"committee_chairs":["Zhang, Chenming Mike"],"committee_members":["Agblevor, Foster Aryi","Van Cott, Kevin E.","Cundiff, John S."],"year":2003,"date_issued":"2003-02-05","date_published":"2003-02-05","updated_at":"2026-07-22T22:19:35Z","subjects":["Lysozyme","Tobacco","Protein Purification","Aqueous Two-phase extraction"],"languages":[],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-02192003-164258"],"render_values":[{"text":"etd-02192003-164258","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/10919/31272","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Zhang, Chenming Mike"]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Agblevor, Foster Aryi","Van Cott, Kevin E.","Cundiff, John S."]},{"key":"dc:contributor.department","label":"Department","values":["Biological Systems Engineering"]},{"key":"dc:creator","label":"Author","values":["Balasubramaniam, Deepa"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2014-03-14T20:31:55Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2014-03-14T20:31:55Z","2004-03-03"]},{"key":"dc:date.issued","label":"Date","values":["2003-02-05"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Tech"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biological Systems Engineering"]},{"key":"thesis:degree_level","label":"Degree Level","values":["masters"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Virginia Polytechnic Institute and State University"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Lysozyme","Tobacco","Protein Purification","Aqueous Two-phase extraction"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["In Copyright"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://rightsstatements.org/vocab/InC/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-02192003-164258"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/10919/31272"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Tobacco 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."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Master of Science"]},{"key":"dc:title","label":"Title","values":["Lysozyme Separation from Tobacco Extract by Aqueous Two-Phase Extraction"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Zhang, Chenming Mike"],"dc:contributor.committeemember":["Agblevor, Foster Aryi","Van Cott, Kevin E.","Cundiff, John S."],"dc:contributor.department":["Biological Systems Engineering"],"dc:creator":["Balasubramaniam, Deepa"],"dc:date.accessioned":["2014-03-14T20:31:55Z"],"dc:date.available":["2014-03-14T20:31:55Z","2004-03-03"],"dc:date.issued":["2003-02-05"],"dc:description.abstract":["Tobacco 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."],"dc:description.degree":["Master of Science"],"dc:identifier.other":["etd-02192003-164258"],"dc:identifier.uri":["http://hdl.handle.net/10919/31272"],"dc:publisher":["Virginia Tech"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:subject":["Lysozyme","Tobacco","Protein Purification","Aqueous Two-phase extraction"],"dc:title":["Lysozyme Separation from Tobacco Extract by Aqueous Two-Phase Extraction"],"dc:type":["Thesis"],"thesis:degree_discipline":["Biological Systems Engineering"],"thesis:degree_level":["masters"],"thesis:degree_name":["Master of Science"],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:19:35Z"}