Advisor(s) - Committee Chair
David Hartman, Lowell Shank, John Reasoner, Robert Farina
Department of Chemistry
Master of Science
Some properties of esterases of the greater wax moth larvae, Galleria mellonella (L.) were examined and the enzyme partially purified. Tris buffer was chosen as the buffer system over veronal buffer. The former causes less hydrolysis of the acetylsalicylic acid. Esterase activities were determined with acetylasalicylic acid in tris buffer (00.05 M, pH 7.85 with 0.1 M EDTA) and with acetyl-p-hydroxybenzoic acid in the same buffer. The enzyme was partially purified by combination of acetone powder preparation, ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration.
The starting enzyme preparation was obtained from aceton powder preparation. The ratio of esterase activity to protein content was increased 1.36-fold by 70% ammonium sulfate precipitation. The ammonium sulfate in the 60-70% saturation range is reproducible with the highest specific activity at 60% saturation. The ammonium sulfate fractionation increased the stability of the enzyme preparation. After 24 hours more than 55% of enzyme activity from the ammonium sulfate precipitate still remained, whereas only about 40% of the enzyme activity from the acetone powder preparation remained.
The pH optimum was found to be 8.5 with acetyl-p-hydroxybenzoic acid as the substrate. The esterase had a linear increase in activity with increasing temperature. At 51°C, the specific activity was 27.60 micromoles/min mg protein in comparison to 12.94 micromoles/min mg protein at 20°C. The kinetic behavior of the enzyme with the different substrates was studied. The Vmax and Km values were 12.5 micromoles/min mg protein and 0.435 millimoles, respectively. The energy of activation with acetyl-p-hydroxybenzoic acid was 4.542 kcal K-1 mol-1.
The esterase activity was inhibited by mercuric chloride and veratrine sulfate. It was activated by the chelating agent, EDTA. The esterase activity was not affected by p-chloromercuribenzoic acid and eserine salicylate.
Juraimi, Salmah, "The Esterases of the Galleria Mellonella Larvae" (1986). Masters Theses & Specialist Projects. Paper 1736.