In Vitro Enzymatic Activity of Apee Esterase with Phospholipids
Location
CSU Ballroom
Start Date
16-4-2013 10:00 AM
End Date
16-4-2013 12:00 PM
Student's Major
Biological Sciences
Student's College
Science, Engineering and Technology
Mentor's Name
Christopher Conlin
Mentor's Department
Biological Sciences
Mentor's College
Science, Engineering and Technology
Description
The apeE gene found in Salmonella typhimirium, a pathogenic gram-negative bacterium that causes infections ranging from mild, self-limiting enterocolitis to systemic typhoid fever. It encodes an outer membrane esterase whose expression is induced by phosphate starvation. Prior studies have shown that ApeE esterase is active against a variety of chromogenic ester substrates and the synthetic lipid analogue Tween80. However, the physiological substrates for the enzyme have not been determined. In order to study the in vitro activity of the ApeE esterase against phospholipids, the amino terminal domain of ApeE, which encodes the esterase activity, was cloned into an expression vector with seven histidine residues attached to the amino terminus. The esterase activity was purified by affinity chromatography using a nickel-chelating resin. Electrophoresis and p- nitrophenyl caprylate (chromogenic esterase) assay were also used to determine the purity and activity of the esterase. Purified ApeE esterase was incubated with the phospholipid phosphotidylcholine, and the extracted products were analysed using thin layer chromatography. Results showed that phosphatidylcholine was indeed hydrolyzed by the enzyme, further supporting the hypothesis that ApeE is used by the bacterium to scavenge phosphate from phospholipids.
In Vitro Enzymatic Activity of Apee Esterase with Phospholipids
CSU Ballroom
The apeE gene found in Salmonella typhimirium, a pathogenic gram-negative bacterium that causes infections ranging from mild, self-limiting enterocolitis to systemic typhoid fever. It encodes an outer membrane esterase whose expression is induced by phosphate starvation. Prior studies have shown that ApeE esterase is active against a variety of chromogenic ester substrates and the synthetic lipid analogue Tween80. However, the physiological substrates for the enzyme have not been determined. In order to study the in vitro activity of the ApeE esterase against phospholipids, the amino terminal domain of ApeE, which encodes the esterase activity, was cloned into an expression vector with seven histidine residues attached to the amino terminus. The esterase activity was purified by affinity chromatography using a nickel-chelating resin. Electrophoresis and p- nitrophenyl caprylate (chromogenic esterase) assay were also used to determine the purity and activity of the esterase. Purified ApeE esterase was incubated with the phospholipid phosphotidylcholine, and the extracted products were analysed using thin layer chromatography. Results showed that phosphatidylcholine was indeed hydrolyzed by the enzyme, further supporting the hypothesis that ApeE is used by the bacterium to scavenge phosphate from phospholipids.
Recommended Citation
Tanvir, Sarah and Chery Vang. "In Vitro Enzymatic Activity of Apee Esterase with Phospholipids." Undergraduate Research Symposium, Mankato, MN, April 16, 2013.
https://cornerstone.lib.mnsu.edu/urs/2013/poster-session-A/2
