Hiroto Saeki
Ritsumeikan University Grad. Sch. Life science, Japan
Title: Enzymatic synthesis of the anti-atherosclerotic dipeptide, Trp-His, using L-amino acid esterase from Pseudomonas aeruginosa PAO1
Biography
Biography: Hiroto Saeki
Abstract
L-Trptophanyl-L-histidine (Trp-His) is the anti-atherosclerotic dipeptide and acts as a vasorelaxant by binding to an extracellular site of Ca2+ channels and regulating intracellular Ca2+ concentration. In addition, Trp-His reduces plaque formation in blood vessels and prevents arteriosclerosis. The dipeptides are generally produced by chemical synthetic methods, but such methods often require the complicated steps and the use of organic solvents which have a negative impact on the environment. L-Amino acid esterase (LAE) can synthesize dipeptides from aminoacyl methyl esters and free amino acids without ATP in one step under mild conditions. In this study, we characterized LAE from Pseudomonas aeruginosa PAO1 (PaLAE) for the synthesis of Trp-His.
The PaLAE gene was inserted into the pCold TEV vector and expressed in Escherichia coli BL21 (DE3). PaLAE was purified by Ni-affinity and gel filtration chromatography with a yield of 35.6% and a purification of 2.37 fold. The apparent molecular mass of the purified enzyme was calculated to be 64 kDa by gel filtration, and the molecular mass of the denatured enzyme was determined to be 66 kDa by SDS-PAGE, indicating that PaLAE was a monomer. The reaction products were measured by HPLC for enzyme activity. PaLAE could synthesize Trp-His using Trp-OMe and L-histidineas. However, PaLAE hydrolyzed Phe-OMe, Tyr-OMe, and Leu-OMe more than Trp-OMe as substrates. These results suggested that PaLAE preferred bulky or hydrophobic amino acid residues.