Wednesday, November 10, 2010
Hall 1 (Salt Palace Convention Center)
A multi-enzyme system involving the catalysis of pyruvate to isobutanol was characterized in this research. This system includes the five enzymes directly active in creating the bio-fuel product and also an additional enzyme necessary for regeneration of NADPH. A poly-Histidine tag was included in the vector to allow for effective purification of the enzymes produced through affinity chromatography. A transfection technique was utilized for incorporation of the plasmid DNA generated into the chosen host cell. Cells were cultured and later disrupted to release over-expressed proteins through the mechanical means of pressure lysis and ultra-sonic disruption. Assays for each protein were used to determine baseline kinetic activity for the individual enzymes. Enzymes were then sequentially mixed to study the kinetics of the multi-enzyme system. Concentration of reaction intermediates and products were determined through the use of HPLC.