Poster Presentation 1B-54

Improved Thermostability of Esterase by Error-prone PCR and Directed Evolution

Yun-Jung Kim,¹ Gi-Sub Choi,¹ Geun-Joong Kim² and Yeon-Woo Ryu^1*

¹Department of Molecular Science and Technology

Ajou University

Suwon, 443-749, Korea

Phone: 82-31-219-2449

Fax: 82-31-216-8777

E-mail: ywryu@ajou.ac.kr

²Institute of Biotechnological Industry

College of Engineering

Inha University

Incheon, 402-751, Korea

Biocatalysts used in various industrial processes must have economical advantages as well as higher substrate specificity. However, enzymes obtained from natural conditions typically have a low thermostability and pH stability, causing them to be unsuitable for manufacturing applications. In many cases, enzymes obtained in normal conditions have property limitations (e.g. substrate spectrum, thermostability). In order to resolve a ketoprofen ethyl ester, which is widely used to relieve pain and reduce inflammation, a directed evolution of an esterase from Pseudomonas fluorescens KCTC 1767 (PFE) was performed. Through ep-PCR and StEP several mutants showed improved thermostability, while enantiomeric excess was either the same or slightly decreased. Also, solvent (e.g. ethanol, butanol, hexane) resistance and surfactant resistance was tested but didn’t show a remarkable result. Sodium dodecyl sulfate in particular did not show any catalytic activity in 0.01% concentration. Optimal pH and pH stability was not changed in our mutation area. DNA sequence of selected mutant showed a few changed amino acids which included an important residue for enzymatic activity.